Disrupting domain-domain interactions is indispensable for EngA-ribosome interactions

    Disrupting domain-domain interactions is indispensable for EngA-ribosome interactions Soneya Majumdar, Abhishek Acharya, Sushil Kumar Tomar, Balaji Prakash PII: DOI: Reference:

S1570-9639(16)30262-X doi:10.1016/j.bbapap.2016.12.005 BBAPAP 39872

To appear in:

BBA - Proteins and Proteomics

Received date: Revised date: Accepted date:

7 October 2016 6 December 2016 10 December 2016

Please cite this article as: Soneya Majumdar, Abhishek Acharya, Sushil Kumar Tomar, Balaji Prakash, Disrupting domain-domain interactions is indispensable for EngA-ribosome interactions, BBA - Proteins and Proteomics (2016), doi:10.1016/j.bbapap.2016.12.005

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ACCEPTED MANUSCRIPT

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ribosome interactions.

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Disrupting domain-domain interactions is indispensable for EngA-

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Soneya Majumdar1, Abhishek Acharya1, Sushil Kumar Tomar1 and Balaji Prakash*2

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Department of Biological Sciences and Bioengineering; Indian Institute of Technology, Kanpur,

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208016, India.

2

570020, India

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Department of Molecular Nutrition, CSIR-Central Food Technological Research Institute, Mysore,

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Running Title: Inter-domain interactions mediate EngA-ribosome association

*To whom correspondence should be addressed: Telephone: 91-821-251-4192. Email: [email protected].

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ACCEPTED MANUSCRIPT Keywords: G-domains, Ribosome biogenesis, EngA, GD1-KH interface, Inter-domain

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regulation.

Abbreviations: Cryo-EM, Cryo Electron Microscope, PTC, Peptidyl Transferase Center,

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MD, Molecular Dynamics, SMD, Steered Molecular Dynamics, COM, Center-of-Mass, WHAM, Weighted Histogram Analysis Method, ITC, Isothermal Titration Calorimetry,

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PMF, Potential of Mean Force.

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ACCEPTED MANUSCRIPT ABSTRACT

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EngA consists of two tandem GTPase-domains - GD1 and GD2 - followed by a KH-domain. EngA was considered to be a 50S assembly factor since it was shown to bind 50S and its

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deletion leads to the accumulation of immature 45S ribosomal subunits. Subsequently, we demonstrated an additional ribosome bound state of EngA bound to 50S, 30S, and 70S. While the former (50S binding) is achieved upon GTP binding at both GD1 and GD2, the

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latter is formed upon GTP hydrolysis at GD1, which is believed to trigger a large

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conformational change in the protein. The present study brings out two key aspects of EngA regulation: First, that distinctly stabilized GD1-KH interfaces allows EngA to exist in

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different ribosome bound states, and second is the importance of these states to ribosome assembly. Our analyses suggest that distinct inter-domain (GD-KH) interfaces are stabilized

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by interactions arising from unique sets of motifs, conserved across EngA homologues, and seem to be mechanistically linked to GTP/GDP binding. By experimentally measuring

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binding affinities for several interface mutants, we show that disrupting the interface

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interactions is necessary to realize EngA-ribosome binding. These findings are also supported by a recent cryo-EM structure of EngA bound to 50S, wherein the GD1-KH interface is completely disrupted leading to an ‘extended’ or ‘open state’ of the protein. Overall, it appears that the transition of EngA from a ‘closed state’ with GD1-KH forming a tight interface, to an ‘open state’ mediates interaction with ribosomal subunits.

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ACCEPTED MANUSCRIPT 1. INTRODUCTION

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GTPases are molecular switches that transit between an active/ON (GTP bound) and inactive/OFF (GDP bound) state to regulate several important cellular processes. Ribosome

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biogenesis is no exception, as six highly conserved subfamilies of GTPases like Era, Obg, YihA, YchF and HflX, regulate various steps in this process [1]. EngA (also known as YphC/Der) is one of the 11 highly conserved GTPases in bacteria [2]. It belongs to the Era

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family of GTPases and is exclusive to the eubacteria [3] and plants [4]. Interestingly, EngA

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comprises two contiguous G-domains (GD1 and GD2) in addition to a KH- like RNA binding domain. Studies suggest that each of these domains are essential for the biological

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activity of this protein [5]. Also, the two G-domains function cooperatively [5] allowing a multifaceted, precise regulation that may be essential for a process as complicated as

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ribosome biogenesis.

EngA is known to be involved in the maturation of 50S ribosomal subunits. E. coli

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cells deficient in EngA show altered ribosome profiles, marked by an increased accumulation of 50S and 30S at the expense of 70S [3]. This is further accompanied by an accumulation of both 23S and 16S rRNA. These 50S ribosomal subunits which accumulate upon EngA depletion are unstable at low Mg2+ concentration and readily dissociate into 40S particles deficient in L9 and L18 ribosomal proteins. Similar effects were noted upon depleting YphC - the EngA homologue in B. subtilis [6]. The aberrant 50S subunits of B. subtilis forms immature 45S particles deficient in three ribosomal proteins L16, L27 and L36 [6]. Surprisingly, it was found that over-expression of EngA rescues the phenotypic defects of the null mutant of rrmJ, which codes for a 23S rRNA methylase that methylates U2552 of the Aloop of an intact 50S subunit [7]. In the absence of this modification, association of 30S and 4

ACCEPTED MANUSCRIPT 50S is compromised, leading to the decrease in 70S levels [8]. Recently, a Cryo Electron Microscopy (Cryo-EM) structure of EngA-50S complex was determined. The structure

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reveals that EngA interacts with the peptidyl transferase center (PTC) of the 50S ribosomal subunit. This induces several conformational changes in the 50S, transforming it to a

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conformation representing an intermediate 45S particle [9]. These studies established that EngA is a late ribosome assembly factor. However, the exact molecular mechanism of EngA

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in ribosome biogenesis remains elusive.

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EngA was hitherto known to be involved in 50S ribosome assembly. However, it has been reported that Salmonella typhimurium EngA interacts with the S7 ribosomal protein

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which constitutes 30S. In this study, the authors suggest that EngA might interact with both 50S and 30S ribosomal subunits at different stages of the assembly process [10]. The

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possibility that EngA might interact with 30S is also corroborated by the observation that it

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co-elutes with 16S rRNA [4,11,12]. In our previous studies, we argued that the role of EngA in ribosome biogenesis must arise from its ability to attain multiple guanine-nucleotide bound

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states, thereby permitting an intricate molecular mechanism. For the first time we identified two distinct, nucleotide-dependent ribosome bound states of EngA; one 50S bound state (when both GD1 and GD2 are in GTP-bound state), and another binding to 30S, 50S and 70S (when GD1 and GD2 are in GDP- and GTP-bound state, respectively) [12]. We rationalized that this is achieved due to the ability of EngA to adopt different conformations in its different nucleotide bound states. The existing crystal structures of EngA homologs - YphC from B. subtilis [13] and Der from T. maritima [14] strengthen this view. In the structure of Der, two phosphates in the nucleotide binding pocket of GD1, occupy positions equivalent to the β and γ phosphates of GTP [14]. Based on this, GD1 was assumed to be in the GTP bound state and hence expected to represent the EngA[GTP:GDP] form (different nucleotide 5

ACCEPTED MANUSCRIPT bound states of EngA are represented within square brackets as nucleotides bound to GD1 and GD2 domains, respectively). The structure of YphC represents the EngA[GDP:GDP]

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state, as both the G domains in this structure are bound to GDP. A comparison of the YphC and Der crystal structure shows that relative orientation of GDP-bound GD2 and KH remains

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unaltered in both the structures [13]. However, GD1 that is connected to GD2 (via a long linker) shows a large conformational change with respect to GD2-KH domains, between its GTP and GDP bound forms [12,13] (Fig. 1A). It was observed that this conformational

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change exposes a positive patch extending across the GD2 and KH domains which might

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facilitate interaction with the negatively charged RNA of the ribosome [13]. Further, in-vitro ribosome interaction assays with domain deletion mutants of EngA revealed that ∆GD1-

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EngA in the GMPPNP state binds to 30S, 50S and 70S while ∆GD1-∆GD2-EngA interacts with 30S ribosomal subunit. This indicates that the 30S binding site lies at the KH-domain,

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and possibly the GD1[GTP] state of EngA restricts the access to this site [12].

Additionally, our studies revealed that EngA fails to bind any ribosomal subunit in

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vitro irrespective of its nucleotide bound state, unless the interactions holding the GD1-KH interface is disrupted [12] (Fig 1A). This led us to speculate that disruption of interactions at this interface triggers a conformational change, in turn facilitating interactions between EngA and the ribosomal subunit. The above inference is also supported by the recent cryo-EM structure of EngA-50S complex, which depicts an extended conformation of EngA interacting with the 50S subunit [9]. Unlike the hitherto known conformations of EngA (‘closed’ conformations based on crystal structures), in this ‘open’ conformation, GD1 and KH do not form an interface (Fig 1B, 1C). The above observation (summarized in Table 1) forms the basis for the current study.

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ACCEPTED MANUSCRIPT Here, we attempt to characterize the two GD1-KH interfaces formed in the two distinct nucleotide bound states of EngA ([GDP:GTP] and [GTP:GTP]), which we presume

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to be essential in mediating EngA-ribosome interaction. Initial structural-bioinformatics analysis of YphC and Der led us to identify distinct motifs that could be responsible for

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stabilizing the two different GD1-KH interfaces in these two conformations of EngA. Mutations at these conserved motifs were anticipated to disrupt the GD1-KH interface. Further, we employed isothermal titration calorimetry (ITC) to study EngA-ribosome binding

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and determined the affinities of these mutants with the various ribosomal subunits. Overall

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the current study establishes the significance of the GD1-KH interface in mediating EngAribosome interaction. Finally, based on the relative affinities of EngA for the ribosomal

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subunits, we suggest a hypothetical model elucidating the probable role of EngA in ribosome

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assembly.

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2. MATERIALS AND METHODS

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2.1 Mutants and protein purification

All the mutants of YphC were generated using overlapping PCR method. YphCD297N

mutant

was

generated

using

mutant

primer

5'-

GTAAACAAATGGAATGCTGTTGACAAAGAT-3' and His-YphC full length as a template.

Double

mutants

-

YphC-Y384A/D297N,

YphC-F398A/D297N,

YphC-

I56A/D297N and YphC-N360A/D297N were generated in similar manner using YphCD297N as a template and 5'-CGTTTGAAAATTTACGCTGCGACTCAAGTGTCG-3', 5'CCAAGCTTCGTTGTGGCTGTAAACGATCCGGAA-3', 5'-TATGATTTTAATTTGGC GGATACGGGCGGTATTG-3' and 5'-GTTCAAACAAACGTCTTAGCTGATGTCATC

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ACCEPTED MANUSCRIPT ATGGAC-3' mutant primers, respectively. In all the cases 5'-CCGCATATGATGGGTA AACCTGTCGTA-3' and 5'-CGCGCGGCCGCTTATTTTCTAGCTCTC-3' primers were

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used as forward and reverse primers to amplify the full amplicon(s). These were digested using NdeI/NotI restriction enzymes and cloned into a pQE2 vector. All mutants of YphC

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were expressed in E. coli DH5α cells and purified as described for His-YphC previously [12].

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2.2 Ribosome purification

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Cells were grown in LB media (HiMedia) till OD600 reached ~0.5 and then harvested by centrifugation and lysed in Buffer C (50 mM Tris-HCl pH 7.5, 10 mM MgCl2 and 50 mM

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NH4Cl) containing His cocktail protease inhibitor (Sigma-Aldrich). All the steps were performed at 4ºC. Cell lysate was then treated with RNase free DNase and subjected to

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centrifugation at 30 x 103 g to obtain the supernatant. This was loaded onto a 8 ml cushion of 34% sucrose prepared in Buffer C followed by a ultra-centrifugation at 200 x 103 g. Pellet of

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crude ribosomes was obtained and washed with Buffer C containing 1 M NH4Cl. For 70S

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ribosome purification, the salt (1M NH4Cl) washed crude ribosomes were loaded (10 A254 units) onto a 35 ml sucrose step gradient (17%, 26%, 35%, 45% 8 ml each) prepared in Buffer C, followed by ultra-centrifugation in Sorvall Sure-Spin 630 rotor (28,000 RPM) for 90 min. 500 µl fractions were collected manually and there absorbance at 254 nm measured. RNA was isolated from each fractions (50 μl taken from 500 μl) using phenol-chloroformisoamyl alcohol method. The RNA was analyzed on a formaldehyde-agarose gel (denaturing gel) and fractions corresponding to 70S (having both 23S and 16S rRNA) were pooled. The 70S ribosomes was pelleted from the pooled fractions through ultra-centrifugation in Sorvall Sure-Spin 630 rotor (28,000 RPM) for 2hrs. The pelleted ribosome was resuspended in Buffer C and stored at -80 0C. For 30S and 50S purification the same protocol as 70S is

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ACCEPTED MANUSCRIPT followed with certain modifications. The crude ribosome is incubated overnight in Buffer E containing 50 mM Tris-HCl pH 7.5, 2mM MgCl2 and 50 mM NH4Cl. It is then loaded (10

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A254 units) onto a sucrose gradient (17%, 26%, 35%, 45% 8ml each) prepared in Buffer E and centrifuged similarly for 2 hrs. Fractions are collected and analyzed as before. The fractions

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corresponding to 30S and 50S are pooled separately, pelleted, resuspended in Buffer E (50 mM Tris-HCl pH 7.5, 2mM MgCl2 and 50 mM NH4Cl) and stored in -800C.

Structural

coordinates

of

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2.3 Structural analysis and sequence alignment

B.subtilis

YphC

(PDB:2HJG),

T.maritima

Der

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(PDB:1MKY) and E.coli EngA (PDB:3J8G) were obtained from PDB and superimposed. These were visually inspected, analyzed and rendered for figures using Chimera [15]. Protein

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sequence of EngA were obtained using PSI-BLAST [16] and redundancy (more than 70%) within the sequences was removed using CD-HIT program [17]. A multiple sequence

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alignment of these was generated using ClustalX program [18]. Figures were prepared using

shown.

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Jalview sequence editor [19] and a representative alignment of 16 protein sequences is

2.4 Homology Modelling

YphC protein sequence was modelled on the Der structure in [GTP:GDP] state (PDB: 1MKY). The protein sequences were aligned using ClustalX [18] and EMBOSS Water webservice from EMBL-EBI website [20]. The alignment was manually checked for errors and was subsequently used as an input for homology modelling. The modeling was performed using Modeller 9.14 [21] which produces a homology model by first deriving a set of spatial

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ACCEPTED MANUSCRIPT restraints from the template and then fitting the query sequence to these spatial restraints as

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best as possible. The top scoring model was used for structural analysis.

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2.5 Umbrella Sampling MD simulations

The model for calculations was B. subtilis YphC (PDB ID: 2HJG). The missing

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flexible regions were modeled using Modeller 9.14 [21]. The top scoring model was

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validated using PROCHECK [22]. Simulations were performed using Gromacs 4.6.7 [23]. The structure was prepared for Umbrella Sampling [24–26]in 2 steps; 1) 120 ns MD to obtain

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equilibrated structures, 2) generation of sampling windows. The protein was placed in a triclinic box solvated with TIP3P [27] explicit water model. The protein parameters were

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derived from the AMBER ff99SB-ILDN parameter set [28]. The system was energyminimized and equilibrated for 2 ns (1 ns each of NVT and NPT equilibration) to obtain a

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system with target conditions of 300 K and 1 bar. For the above steps, Berendsen weak

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coupling [29] algorithm was used for temperature and pressure coupling. 120 ns of MD was performed on the relaxed protein. The temperature and pressure was controlled using NoseHoover thermostat [30,31] and Parrinello-Rahman [32,33] barostat respectively to ensure a correct canonical ensemble. The equilibrated final structure was edited to delete amino acids

173 and 174 that lie in the flexible linker region between GD1 and GD2. The ends produced by the deletion were capped by ACE and NME respectively at C and N terminal to generate uncharged ends. This break was introduced to allow for pulling of GD1 away from GD2-KH domains. The system was equilibrated for an additional 1ns before proceeding to generation of sampling windows. Sampling windows were obtained by performing Steered Molecular Dynamics (SMD) simulations [34]. For this step, the protein was appropriately placed and 10

ACCEPTED MANUSCRIPT oriented in a rectangular box with dimensions 16 nm x10 nm x10 nm. The box dimensions were chosen to be longer along the X-axis to allow for the COM-pulling of GD1 along this

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direction while maintaining the Periodic Boundary Condition. The pulling simulations were performed with GD2-KH domains as the reference group and GD1 as the pull-group. The

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GD1 domain was pulled away from GD2-KH with a pull rate of 0.01 nm ps-1 for 500 ns. The force constant applied for pulling was 1000 kJ mol-1 nm-2. The GD1 COM was pulled apart along the x-axis to a maximum distance of 4.7 nm from GD2-KH domains. From this

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trajectory, snapshots were extracted to serve as starting coordinates for umbrella sampling.

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The sampling windows were selected according to an asymmetric distribution scheme as described previously [35]. This resulted in 25 sampling windows which were simulated for

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10 ns each. The results were analyzed by Weighted Histogram Analysis Method (WHAM)

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[36] using the g_wham utility of Gromacs.

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2.6 Fluorescent Nucleotide Binding

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N-methyl-3'-O-anthranoyl (mant) labelled nucleotides were employed to carry out Fluorescent nucleotide binding studies. 4.0 µM of the respective protein and 0.4 µM mantnucleotides (mant-GDP/mant-XDP) were incubated in Tris-HCl Buffer 50 mM (pH 7.0), containing 200 mM KCl, and 2 mM MgCl2 at room temperature for 10min. Emission profile of the samples was monitored from 400 to 600nm after excitation at a wavelength of 355nm. The slit widths for excitation and emission were kept at 10nm and the scan speed was 300nm/min. The curves plotted are the average of three consecutive scans.

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ACCEPTED MANUSCRIPT 2.7 Isothermal Titration Calorimetry (ITC)

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ITC was performed to quantify the affinity of ribosome-EngA interaction in different nucleotide bound states of the mutant proteins using ITC200. The experimental parameters

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maintained for the purpose was adapted from Recht et. al., 2008, with certain modifications [37]. Briefly, the protein and the ribosome was dialysed in a buffer containing Tris 25mM pH 7.5, 2mM MgCl2 and KCl 330mM (50S and 30S) or Tris 25mM pH 7.5, KCl 330mM and

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MgCl2 10mM (70S). Thereafter, appropriate nucleotides (GDP/GMPPNP/XMPPNP) at a

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concentration of 0.5mM was added to both the protein and the ribosome, based on the mutant under study and the required nucleotide bound state. The sample cell was filled with 280μl of 0.5-1μM of ribosome (having nucleotide) and the syringe with 10μM -15μM of protein

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(having nucleotide). 15-20 injections of 2μl protein was added to the ribosome at intervals of

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4min with stirring at 400rpm. Microcal Origin 7 was used to analyse the data. The heat of dilution was determined by titrating the protein (with 0.5mM of respective nucleotide) into

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buffer (with 0.5mM of respective nucleotide). An additional control experiment was carried

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out in which buffer (with 0.5mM of respective nucleotide ) was titrated into 1μM of ribosome (with 0.5mM of respective nucleotide) to ensure that the heat change obtained was not a result of disassembly of the ribosomal complex. The data best fitted to a “one-set-of-site” binding model to obtain the binding constant (K) and stoichiometry (n). The c-values (K ×protein conc.× n) of all the ITC data was in the acceptable range of 1-1000 [38]. The dissociation constant (Kd) was calculated as 1/K. All the experiments were performed in duplicates.

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ACCEPTED MANUSCRIPT 3. RESULTS

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3.1 Comparison of three distinct conformations of EngA.

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EngA homologues share significant sequence identity and conserve the overall domain architecture. For instance, YphC (from B. subtilis) shares ~39% sequence identity with Der (from T. maritima) and ~38% with EngA (from E.coli) (Fig. 2 and 3). In EngA

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structures, nucleotide binding pockets of GD1 and GD2 are ~55Å apart and located at the

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opposite ends of the molecule. Structural comparison of Der and YphC crystal structures representing the two distinct nucleotide bound states of EngA shows that GD1 undergoes an

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enormous conformational change (~60 Å) between the GTP and GDP bound states, (Fig.1A). This movement of GD1, exposes a new GD1-KH interface and relocates its nucleotide-

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binding pocket with respect to the KH domain. In EngA[GTP:GDP] state, the nucleotide binding pocket of GD1 is ~8 Å away from KH, while in EngA[GDP:GDP] state, it is ~25 Å

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away. For ease of presentation, we term the GD1-KH interface in EngA[GDP:GDP] state as

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GD1GDP-KH and that in EngA[GTP:GDP] state, as GD1GTP-KH (see red and green broken lines, Fig. 1A). Depending on its GTP or GDP bound states, the two interfaces receive contributions from different regions of GD1, but the contribution from KH is largely the same. In a previous work on EngA, we showed that the GD1-KH interface of the protein needs to be disrupted to enable its interaction with the ribosome in vitro [12]. This is supported by the recent cryo-EM structure of EngA from E.coli wherein the protein acquires an extended ‘open’ conformation - the GD1 and KH no longer form an interface [9] (Fig. 1B,C). In order to understand how the open conformation which facilitates the binding of EngA to ribosomes is achieved, we began analyzing the structures of EngA homologues (YphC and Der) in which the GD1 and KH form an interface. This analysis revealed two

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ACCEPTED MANUSCRIPT distinct interfaces, formed when GD1 is either bound to GTP or GDP, which are presented

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below.

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3.2 Identification of conserved motifs at the GD1GDP-KH interface

The GD1GDP-KH interface (Fig. 1A) seen in the EngA[GDP:GDP] state, i.e YphC structure, involves interactions from a) a negatively charged α4 helix of GD1 and the region

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preceding it; and b) β-sheets β14 and β15 of the KH domain (Fig. 2B). Here, the overall

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negative charge on the surface of GD1 is stabilized by the positively charged face of the KH domain. In addition, a strong hydrophobic stabilization is also seen at this interface, and the

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residues involved in stabilizing the interfaces are highly conserved (Fig. 2A).

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In the GD1GDP-KH interface, Asp132 and Tyr134 from the conserved

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DFYSLG137

region (α4 helix) of GD1, interact with two conserved regions of the KH domain, i.e. Tyr384, 381

KIYYATQ387 region (β14) and Val397, Phe398 of the

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Thr386 of

395

FVVFVN400 region

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(β15). These regions stabilizing the interface appear to be highly conserved (Fig 2 A, B). Based on the conservation pattern, it is possible to describe consensus sequence motifs M1, M2 and M3 tabulated in Table 2. Overall, our analysis of the GD1GDP-KH interface revealed three consensus sequence motifs M1, M2 and M3; a number of conserved residues from these motifs contribute to inter-domain interactions between GD1 and KH (Fig 5A).

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ACCEPTED MANUSCRIPT 3.3 Identification of conserved motifs at the GD1GTP-KH interface

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Similar to the GD1GDP-KH interface, the GD1GTP-KH interface seen in the EngA[GTP:GDP] state (Fig. 1A), is stabilized by residues provided by a) the switch II region, and helix α8 of

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GD1; and b) helix α12, strand β14 and β15 of the KH domain (Fig. 3A, 3B). In order to compare the two distinct conformations of EngA from the same homologue, a homology model of YphC was generated employing the crystal structure of Der (EngA[GTP:GDP]

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state) as template [see section 2.4]. As already mentioned, the YphC shows a sequence

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identity of ~39% with Der sequence which indicates the suitability of the Der structure as a template. In this model of YphC in the [GTP:GDP] state, Ile-56, Asp-57 and Ile-61 from

provide

charged

and

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switch II region (55LIDTGGI61), and Ile-72 and Ala-79 from helix α8 (72IRQQAEIAM80) hydrophobic

interactions

to

Asn-360

from

helix

α12

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(353RVQTNVLN360), Tyr-384 from β14 region (381KIYYATQ387) and Phe398 from β15 region (395FVVFVN400) of the KH domain. Similar to the GD1GDP-KH interface shown in

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figure 2, sequence alignments allow the identification of consensus sequence motifs tabulated

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in Table 2. From GD1, motifs M4 and M5 provide residues that form stabilizing interactions at the interface. Similarly, from the KH domain, the motif M2, M3 and M6 motifs contribute residues to stabilize this interface (Fig. 3A, 3B and Fig. 5B).

3.4 Stability of the GD1-KH interfaces

In order to evaluate the stability of the GD1-KH interfaces and identify the interactions primarily involved in its stability, we performed Molecular Dynamics (MD) studies using the YphC structure as a candidate. The free energy change accompanying the binding of two molecular entities can be obtained by calculating the Potential of Mean Force

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ACCEPTED MANUSCRIPT (PMF). However, reliable estimation of ΔG values require extensive sampling of the phase space, which is not possible with limited computational time and resources. One of the

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techniques employed for extensive sampling of data is Umbrella Sampling [24–26]. Specifically, this method involves the generation of sampling windows (using pulling

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simulations or Steered Molecular Dynamics) that are essentially snapshots of the coordinate changes as the two interacting entities - one reference molecule and the other molecule which is pulled away from the reference molecule - are pulled apart by a constant force along a

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reaction coordinate. For each of the snapshots, an independent simulation is conducted

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keeping the pulled molecule at a constant distance from the reference molecule using a restraining potential. This allows the interacting molecules in each of the windows to sample

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the phase space for an appropriate time without significant changes in their Center-of-Mass (COM) distances. The end result is the generation of an ensemble of structures along the

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specified reaction coordinates. The PMF for each of the windows is calculated and energy values are assembled so as to obtain a continuous function. This represents the PMF along

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the reaction coordinate [35].

Here, for simplicity, we have chosen the reaction coordinate ξ as the dissociation of GD1 from KH domain linearly along one direction (Supplementary Fig. S1). Steered Molecular Dynamics (SMD) simulations were conducted on YphC to pull GD1 along this reaction coordinate, ξ. This method, in addition to providing snapshots for Umbrella Sampling, also allows the generation of the dissociation pathway and the time evolution of various interaction involved. However, there are certain implications of the choice of a simplified reaction coordinate, as we have chosen in our case. The dissociation of GD1 from GD2-KH is not expected to occur along a straight reaction coordinate because a) the movement of the GD1 domain with respect to GD2-KH is constrained by the linker and b)

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ACCEPTED MANUSCRIPT based on the positions of the three interfaces seen in EngA structures, it can be inferred that the movement of GD1 domain from one state to another must be governed by complex

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rotational and translational forces. Therefore, the dissociation pathway of GD1 along a straight line as such may not be biologically significant to merit a detailed analysis. However,

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it can provide an approximate idea of the interactions involved and the stability of the studied interface. Below we have described the key features of this simplistic dissociation pathway, in order to highlight the important residues that might be involved in stabilizing the GD1-KH

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interface.

The Force vs. Time plot obtained from the SMD simulation shows a linear increase in

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pulling force till 175 ps, after which, there is a disruption of interactions holding the two domains, as indicated by a decrease in the pulling force (Fig. 4A). However, a visual

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inspection of the trajectory reveals, the GD1GDP-KH interface starts to destabilize only after 184 ps. A decrease in the pulling force after 175 ps, coupled with the observed delay in

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destabilization of GD1 and KH interface suggests the involvement of additional interactions

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that are disrupted prior to the destabilization event. Further analysis revealed the participation of residues in the GD1-GD2 linker (residues 168-174) in providing additional interactions that stabilize both GD1 and GD2 (Fig. 4B). The linker region supplies residues that bridge GD1 and GD2; Tyr171 and Glu168 are separately involved in stable interactions with Asp215 and Arg211 of GD2 domain, while Lys170 forms a salt-bridge with Asp84 of GD1. After 184 ps, the interactions at the GD1-KH interface get completely disrupted within the next 16 ps. By ~260 ps, the GD1 domain is completely solvent exposed and its interaction with the GD2-KH domain is suspended. The SMD trajectory can be visualized in a video available online as supplementary material. Figure 4C depicts the interactions at the GD1GDPKH interface. The two domains are primarily held by electrostatic interactions involving a

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ACCEPTED MANUSCRIPT number of interface residues. Most prominently, at the core of the interface lie three aromatic residues, Tyr131, Tyr384 and Phe398 that provide additional π-stacking interactions (Fig.

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4C). During the course of the dissociation pathway observed in the SMD, these stacking interactions were seen to be the most stable, lasting the full duration (till 184 ps) of the time

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GD1-KH interface was intact. Additionally, Tyr134 stabilizes Glu140 that forms a salt-bridge with Arg433. Thr386 is involved in a hydrogen bond with backbone oxygen of Tyr134. SMD simulations were also run at slower rates (0.005 and 0.001 nm ps-1) to examine for artifacts

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that might be introduced due to different pull-rates. Although, the time and maximum force

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required for dissociation varied due to the different pull-rates used, the dissociation pathway observed was essentially the same in each case, with similar Force vs Time plots (Supplementary Figure S2). Also, a comparison of the overall shape of the Force vs Time

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plot in case of slowest pull-rate used (0.001 nm ps-1; blue plot in Supplementary Figure S2)

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with that of the curve in case of the fastest pull rate (0.01 nm ps-1; see Figure 4A) gives a better perspective of this similarity. This justifies the faster pull-rate (0.01 nm ps-1) we used

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for fast data collection, at the same time ensuring the reliability of the results. The 25

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windows (trajectory snapshots), extracted at appropriate intervals, (see section 2.5) were simulated and analyzed to obtain the Potential of Mean Force (PMF) curve for the dissociation of GD1 from GD2-KH (Supplementary Figure S3). The ΔG of binding was estimated to be 17.2 ± 0.6 kcal mol-1, suggesting that the interaction at the GD1-KH is significantly stable. To conclude, the simulation studies predicted residues Tyr131, Tyr384 and Phe398, along with Tyr134, as the most prominent interactions stabilizing the GD1GDPKH interface. Structural analysis reveals that two of these residues Tyr384 and Phe398 also stabilize the GD1GTP-KH interface (Fig. 5B). Additionally, these simulations seem to indicate that the interface is significantly stable. Hence, it may be inferred that upon disrupting these

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ACCEPTED MANUSCRIPT key interactions, the stability of the GD1-KH interface would be compromised and an open

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conformation could be achieved.

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3.5 Destabilizing the GD1-KH interface facilitates EngA-ribosome interaction.

In an earlier work, we found that EngA-ribosome binding as observed in assays using EngA over-expressed cell lysates (termed ‘in vivo’assays for convenience), could not be

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achieved in reconstituted assays where the individual components were purified separately

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(termed ‘in vitro’ ribosome interaction assays for convenience) [12]. However, the presence of mild urea in these in vitro assays or the employment of Y134A mutant of YphC, restored

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the anticipated interactions, i.e. with the 50S subunits in EngA[GTP:GTP] state and with 30S, 50S and 70S in the EngA[GDP:GTP] state [12]. Y134 is presented by the sequence motif DFYSLG137 of GD1 and contributes to the GD1GDP-KH interface. We reasoned that

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employing a Y134A mutant of EngA or mild urea with WT EngA, compromises interactions

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ribosome[12].

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at GD1-KH interface allowing the enzyme to attain a conformation suitable to bind the

In the current study, we assessed EngA-ribosome interactions using purified components in vitro. For this, YphC (EngA homolog from B. subtilis) and its mutants for interface residues and ribosomes from B. subtilis were purified. It was expected that disrupting the GD1GDP-KH and the GD1GTP-KH interface would have a similar effect, as observed in case of the Y134A mutant i.e. restoration of YphC-ribosome interactions. Firstly, all mutants were created with a YphC-D297N background. This is because D297 of the G4 motif (NKxD) of GD2, when mutated to N, switches nucleotide specificity of GD2 from guanine to xanthine, Previously, we showed that while D297N mutants bind Xanthine

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ACCEPTED MANUSCRIPT nucleotides at GD2, GD1 that does not carry a similar mutation continues to bind guanine nucleotides [12,39]. In this manner, it was possible to isolate the nucleotide specificities of

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the two G-domains. Fluorescent nucleotide binding assays confirmed that YphC-D297N binds nucleotides as anticipated and significant binding to both of the fluorescent nucleotides,

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mant-GDP and mant-XDP is observed (Fig. 5C). Also, all the mutants generated in the background of YphC-D297N displayed significant nucleotide binding similar to the D297N mutant, indicating that the mutations at the interface do not alter nucleotide binding (Fig. 5D-

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G). Therefore by employing D to N mutants, and by simultaneously using xanthine and

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guanine nucleotides, it was possible to obtain the EngA[GTP:GTP] or EngA[GDP:GTP]

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states.

Ribosome-protein interaction studies usually employ co-sedimentation or micro-

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filtration based assays (Supplementary methods) wherein the ribosome-protein complex is eventually detected using western-blot analysis. These assays are qualitative in nature, which

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only indicate whether or not the proteins bind the ribosome. It fails to provide a quantitative

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value to the affinities of the proteins for the ribosomal subunits. Therefore, we employed Isothermal Titration Calorimetry (ITC) to quantitate the affinities of the YphC interface mutants for 30S, 50S and 70S ribosomal subunits. ITC has been previously employed to determine affinities of protein for ligands and to quantitate protein-protein interactions. For this, 1μM of ribosome (30S or 50S or 70S), taken in the sample cell, is titrated with 10-20 μM of the mutant proteins until saturation is achieved (see section 2.7). The ITC profiles obtained fitted best to a “one-set-of-site model” while analyzing with the Microcal Origin 7 software. The stoichiometry was nearly 1:1 and the c- values were in the permissible range of 1-1000 [38] suggesting that the binding constant, K derived from the isotherms were reliable.

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ACCEPTED MANUSCRIPT The dissociation constant, Kd was calculated as 1/K. All the parameters determined from the

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ITC experiments are tabulated in Table 3.

Firstly, we observed that both YphC wild type and YphC-D297N mutant failed to

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interact with the ribosomal subunits (a representative ITC profile for YphC wild type and YphC-D297N mutant with 50S ribosomal subunit in the GMPPNP:GMPPNP state is shown in figure 6). The mutants, YphC-Y134A/D297N from the 381

DFYSLG137 motif, YphC-

KIYYATQ387 and YphC-F398A/D297N from the region

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Y384A/D297N from the region

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FVVFVN400 of KH bind to only 50S in GMPPNP:XMPPNP state (panel A in Fig. 7-9 and

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395

representative ITC curves for YphC-Y134A/D297N in Supplementary figure 5). Notably, all

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the mutants show binding affinity for 50S, 30S and 70S in GDP:XMPPNP state (panel B-D in Fig. 7-9). However, ITC experiments with two other interface mutants YphC-

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N360A/D297N and YphC-I56A/D297N indicated no specific interaction with the ribosomal subunits (Supplementary figure 6). Hence, it is evident that the ribosome binding behavior is

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specific to YphC mutants for some of the interface residues. This also corroborates with our

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predictions from MD simulation studies where Y134, Y384 and F398 were identified as residues crucial for stabilizing the GD1-KH interface. Further, these results are also in tune with the ribosome-EngA interaction studies performed using co-sedimentation and microfiltration assays (Supplementary figure 4). Overall, the ribosome interaction studies reveal that Y134 from α4 (M1) stabilizing the GD1GDP-KH interface and two other residues Y384 from β14 strand (M2) and F398 from β15 strand (M3) stabilizing both GD1GDP-KH and GD1GTP-KH interface when mutated, are capable of restoring nucleotide dependent interactions with ribosome, in the in vitro assays conducted using purified components.

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ACCEPTED MANUSCRIPT Further, the affinities (based on Kd values in Table 3) of all the mutants, YphCY134A/D297N, YphC-Y384A/D297N and YphC-F398A/D297N is comparatively more

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(towards the 50S subunit) in the GMPPNP:XMPPNP state, and is lowered (towards 50S and 30S) in the GDP:XMPPNP state; further the affinities are least for the 70S ribosome in this

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state (Table 3). Also, we observed that the tyrosine mutants, YphC-Y134A/D297N and YphC-Y384A/D297N show a higher affinity for the 50S and 30S subunits in GMPPNP:XMPPNP and GDP:XMPPNP state (Fig. 7 and Fig. 8) compared to the F398A

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mutant, YphC-F398A/D297N (Fig. 9) – which exhibits a higher affinity towards 70S

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ribosomes (Table3).

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3.6 The importance of GD1-KH interface revealed by the cryo-EM structure of

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EngA-50S.

Based on a recent cryo-EM structure of EngA bound to the 50S ribosomal subunit, a

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number of residues were identified to be critical for EngA-50S interaction [9]. Upon mutation

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of these residues, it was expected that EngA interaction with the 50S ribosome would be compromised. However, only three of the identified residues H147, R149 and E271 when mutated, completely abolished EngA-ribosome binding [9]. We carefully compared the above structure with those in the ‘closed’ conformations to understand whether these residues are similarly positioned and available for interactions with the ribosome.

H147 is highly conserved across species while R149 is not. As shown in Fig. 2A, R149 is substituted by L150 in YphC and I151 in Der. In the E.coli EngA structure, H147 lies in a loop between β6 and α5 of GD1 and interacts with G2286 at the P-site of 23S rRNA (Fig. 10A). Based on a superimposition of the GD1 domains of YphC, Der (in closed

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ACCEPTED MANUSCRIPT conformation) and E.coli EngA, (open conformation) it appears that, in YphC and Der structures, this histidine is not in a conformation suitable to promote interactions with the 23S

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rRNA (Fig. 10A). While, explaining this anomaly requires rigorous experimental studies, analysis of the available structures highlight a few important observations whose relevance to

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ribosome binding can be debated.

First of all, inspection of the loop region comprising the conserved His (hereafter

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called H-loop) in E.coli EngA (residues 143-150), YphC (residues 144-151) and Der reveals

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that E.coli EngA contains two glycine (G148 and G150) and YphC contains three (G146, G149 and G151). The corresponding H-loop in Der (residues 145-151) however does not

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contain any glycine. Further, in E.coli EngA, the H-loop (143-150) interacts with the NKXD motif (119-122) of the G-domain (Fig. 10D). Additionally, the H-loop and the NKXD motif

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are connected via the M1 motif (132-137). Likewise, in YphC and Der too, we find that the H-loop receives interactions from the NKXD motif (Fig. 10B and 10C); here too the M1

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motif forms the connection between the H-loop and NKXD motif. This appears important

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considering that the M1 motif in YphC, forms a part of the GD1GDP-KH interface.

Another highly conserved residue critical for EngA-50S interaction is E271 [9]. E271K mutant of E.coli EngA fails to interact with the 50S ribosomal subunit [9]. However, we could not identify any interaction between E271 and the 50S ribosomal subunit. In a previous study, it was shown that E271K mutant of E.coli EngA also exhibits poor GTP hydrolysis by the GD2 domain (0.2min-1) [40]. Since the GTPase activity was not completely abolished, it was concluded that the E271K mutant affects the turnover of the enzyme i.e. GDP to GTP cycling [40]. We had previously shown that GTP binding at GD2 is a minimal requirement for interaction with any ribosomal subunit. Thus, compromised efficiency in

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ACCEPTED MANUSCRIPT GDP to GTP exchange may explain the inability of the E271K mutant to interact with the

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50S.

Another residue found important in our analysis is a highly conserved Y437, of EngA,

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which interacts with U2593 and C2594 of 23S rRNA. U2593 and C2594 (Fig. 10E) is a part of a stem-loop in the peptidyl transferase center (PTC) and hence this interaction could be critical. A superimposition of Der and E.coli EngA reveals that F414, the residue

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corresponding to Y437 of E.coli EngA seems to be stabilized in a flipped position by a

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conserved V372 from α12 of the Der KH domain. This region, i.e. α12 (M6) forms the

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GD1GTP-KH interface with M5 from α8 (Fig. 10E).

Collectively, these observations point towards a mechanistic link between the

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GTP/GDP binding site, the interface motifs and the conformation of residues that mediate EngA- 23S rRNA interaction. Structural evidence indicates that GD1-KH interface motifs

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may be critical to ribosome interactions; however, elucidating the detailed mechanism and

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understanding their significance necessitate further experimental studies.

4. DISCUSSION

EngA is a conserved and essential ribosome assembly factor in bacteria. It is known to participate in the maturation of 50S ribosomal subunit [3]. It is a multi-domain protein; typically for multi-domain proteins, inter-domain communication at domain boundaries is important in regulating their function. This work brings out two different aspects of regulation by EngA – first is the importance of inter-domain (GD-KH) communication that

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ACCEPTED MANUSCRIPT allows EngA to adopt distinct ribosome bound states, and the second concerns the importance

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of these ribosome bound states to ribosome assembly.

4.1 The importance of domain-domain interactions in facilitating nucleotide dependent

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conformational change.

Previously, in in vitro EngA-ribosome interaction assays, we found that EngA fails to

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interact with ribosome unless treated with mild concentrations of urea [12]. Based on an

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examination of domain-domain interfaces in the available crystal structures of EngA, we speculated that the GD1-KH interface of EngA is critical in facilitating ribosome binding. In

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the current study, by analyzing the GD1-KH interfaces seen in the two different conformations adopted by EngA in its GDP:GDP and GTP:GDP states, we identified key

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residues that distinctly stabilize the two conformations.

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Our analysis revealed that interactions between a negatively charged region of GD1

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and a positively charged surface of KH domain, stabilizing the GD1GDP-KH interface, are well conserved among EngA homologues (Fig. 2). Conserved hydrophobic residues are buried at the interface between the two domains that further strengthen the interactions. This explained why urea could restore EngA-ribosome binding in vitro. Three motifs, namely M1, M2 and M3 (Table 2) stabilizing GD1GDP-KH interface (i.e. EngA[GDP:GTP] state) were identified based on structural analysis. Similarly, at the GD1GTP-KH interface (i.e. EngA[GTP:GTP] state), interactions between switch II region of GD1 and α12, and β14 of the KH domain participate in stabilizing the interface (Fig. 3). The motifs stabilizing GD1GTP-KH interface are M2, M3, M4, M5, and M6 (Table 2, Fig. 3A). In all of these, several residues seem to provide hydrophobic interactions or make direct contacts with each

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ACCEPTED MANUSCRIPT other via H-bonds or salt bridges. Altogether, strong interactions seem to fasten GD1 and KH together, both at GD1GTP-KH and GD1GDP-KH interfaces. Overall these motifs are important

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in (un)fastening the interfaces and allow domain movements that facilitate transition between the ‘open state’ (the one bound to 50S) and the ‘closed state’ (the unbound/free form) of

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EngA.

A previous study on E.coli EngA reports that while WT EngA rescues the lethal

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mutant ∆rrmJ of E.coli, G414R and G424D mutants of EngA do not and they fail to interact

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with the ribosomes [5]. Surprisingly, the cryo-EM structure of E.coli EngA-50S complex reveals that neither of these residues contribute to any interaction with the 50S ribosomes.

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Incidentally, they are present at GD1-KH interface and a part of the M2 and M3 motifs, respectively (Fig. 2A). Hence, it is plausible that the mutations, G414R and G424D, may

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further stabilize the GD1-KH interface and thereby abolish the interaction with 50S by

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restricting the domain movement that allows the transition from a ‘closed’ to an ‘open’ state.

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MD simulation studies performed here elucidated that the identified motifs indeed contribute to the stabilization of the GD1-KH interface. In particular, it suggested that Tyr131, Tyr384, Phe398 and Tyr134 are the most prominent residues stabilizing the interface. These residues when mutated were indeed found to be important for stabilizing the interfaces and thereby facilitate ribosome binding.

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ACCEPTED MANUSCRIPT 4.2 Mechanistic link between the interfaces, the GTP binding site and ribosome binding

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elements:

While this work was in progress, a cryo-EM structure of E.coli EngA-50S complex

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was reported [9]. In this structure, the GD1 and KH domains of the protein are positioned wide apart (i.e. absence of GD1-KH interface) to adopt an ‘open’ conformation upon interaction with the ribosome (Fig. 1A-C). In the light of this new structure we concluded

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that, possibly the GD1-KH interface interactions hold EngA in a ‘closed’ conformation while

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disruption of this interface allows the protein to acquire an ‘open’ extended conformation. We further analyzed the availability of the critical residues interacting with the 50S

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ribosomes. We identified two highly conserved residues in E.coli EngA, H147 (of the Hloop, discussed above) and Y437, which interact with the PTC of 23S rRNA. The structural

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analyses suggest that these two residues may be mechanistically linked to the NKXD motif of the guanine nucleotide binding site and the interface motifs; a cross-talk between these

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structural elements may explain nucleotide dependent regulation of EngA-ribosome

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interactions (Fig. 10).

In the cellular context, it may be suggested that an unknown factor(s) assists the disruption of the aforesaid interactions at the GD1-KH interface; this coupled with the GTP/GDP state of GD1 and GD2 may trigger (or suspend) EngA-ribosome interactions. Such an unknown factor could either be another ribosome assembly factor or a small molecule or any effector, working together with EngA to complete ribosome assembly. In our experiments using purified components, it appears that we have replaced this effect by generating selective mutations at these interfaces. These mutants would allow making stable EngA-ribosome complexes and offer further possibilities to elucidate the molecular function

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ACCEPTED MANUSCRIPT of EngA in ribosome biogenesis. Most importantly, as EngA is uniquely conserved in prokaryotes, these interfaces could be potential targets for the design of specific inhibitors.

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These inhibitors may hinder inter-domain regulation and inhibit appropriate conformations necessary for the biological function of EngA. The interfaces GD1GTP-KH and GD1GDP-KH,

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can be analyzed in detail in a pathogenic (bacterial) EngA homologue, to augment the specificity of the designed inhibitor.

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4.3 Significance of Nucleotide bound states in Ribosome binding.

We performed ITC experiments to quantify the affinities (Kd) of the interface mutants

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for the ribosome. We obtained a Kd of about 0.1μM for EngA (interface mutants) towards 50S ribosomes, but did not find any interactions with WT EngA. In contrast, in a previous

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study, Zhang et al., find that WT EngA binds to 50S ribosomal subunits. In this study, they use ~1.8μM EngA [9], which is clearly in excess of the cellular levels of EngA at any given

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condition. This might explain the anomaly.

We therefore chose not to use very high

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concentrations of EngA; in the ITC experiments, the first injection which would yield a final concentration of 71 nM of EngA (i.e. 2l of 10uM protein added to 280l of ribosome) exhibits significant heat change indicating an interaction of EngA with ribosomes. Also, in the ribosome binding assays (supplementary method) we used 100-200 pmol of protein for 180 pmol of 50S (1 A.U260 ~ 36 pmol) while in the previous study by Zhang et. al., 2014, 600 pmol of protein was used for 30pmol of ribosome. Therefore, the interactions with ribosomes indeed appear to depend on the interactions at the interface; suggesting that the disruption/weakening of the GD1-KH interface is a prerequisite for increased affinity of EngA towards ribosomes.

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ACCEPTED MANUSCRIPT YphC mutants exhibited high affinity for 50S ribosomal subunits in the GMPPNP:XMPPNP state (Kd~80 nM). In contrast, in the GDP:XMPPNP state, while the

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affinity for 50S is similar, they additionally bind 30S (and 70S too, albeit weakly). Based on a comparison of these affinities for the different ribosomal subunits in these two states, we

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present a plausible model suggesting a role for EngA in ribosome assembly (Fig. 11). Previous studies clearly suggest that EngA depleted cells accumulate immature pre-50S particles [3,5]. These particles are sensitive to Mg2+ concentrations - at low Mg2+ levels the

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complex collapses, shedding off various ribosomal proteins, while at higher levels of Mg2+

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the complex remains intact [3,5]. Further, from the cryo-EM structure of EngA-50S complex, it was observed that the interactions of EngA with the 23S rRNA at the PTC involves highly

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conserved residues- H147 and Y437. From this structure, EngA interacts with rRNA and the ribosomal proteins L1 and L2, which are primary ribosomal proteins directly recruited to 23S

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rRNA. Since no interactions were found with L16, L27, L33 and L36 (secondary and tertiary ribosomal proteins), it appears that EngA may not have a direct role in assembling ribosomal

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proteins onto the 23S rRNA, and the loss of these proteins (L16, L27 and L36) reported in

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previous studies in the EngA deletion strains [6] could be a result of an unstable pre-50S ribosomal complex. In the study by Zhang et. al., as well, it was proposed that the binding of EngA to the 50S ribosomal subunit induces conformational changes in 50S such that it acquires a conformation resembling a pre-50S unit; in which several rRNA helices are displaced and the ribosomal protein L33 is depleted. Further, they suggested that binding of EngA to 50S would subsequently inhibit association with 30S.

Overall, the above studies

indicate that EngA might be involved in the final conformational stabilization of the pre-50S complex.

29

ACCEPTED MANUSCRIPT EngA has also been reported to co-elute with 16S rRNA. In a previous study from our lab, we reported that EngA interacts with 30S in GDP:GTP bound state. Further, a study on

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Salmonella typhimurium indicates that EngA interacts with the 30S ribosomal proteins S7 [10]. Interestingly, S7 lies on the 30S ribosomal subunit interacting with the 50S. In the

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current study, the ITC experiments revealed that EngA specifically interacts with 30S with significant affinity and with 70S with a comparatively lower affinity (panel C of Fig. 7-9,

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Table 3).

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4.4 A plausible model for the role of EngA in ribosome assembly.

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We presume that EngA binds to a pre-50S complex in the GTP:GTP state (Fig. 11, [9]). Upon hydrolysis of GTP at GD1, it achieves a GDP:GTP state and a conformational

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change with an altered position of GD1 relative to the rest of the protein, occurs. Zhang et al. too suggest that GD1 occupies the same position as L33 [9]. Taken together, we speculate

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that the conformational change in EngA would allow L33 to associate with the pre-50S; and

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thereby allow the pre-50S complex to mature into the fully formed 50S. However, our results suggest that EngA (both GTP:GTP and GDP:GTP) binds strongly to matured 50S (panel A and B of Fig.7-9, Table. 3), suggesting that it continues to bind 50S even after maturation. It may be that the release of EngA from the 50S complex requires GTP hydrolysis at GD2 domain, which perhaps induces a different conformation that triggers the release (Fig. 11).

We also find that the affinity of EngA to 50S is relatively unchanged between the GTP:GTP and GDP:GTP states. However, there is binding to 30S and 70S in the GDP:GTP state, but not in the GTP:GTP state (supplementary figures 4 and 5 for interaction of the mutants with 30S and 70S; Tomar et al; 2009 for interactions of the Y134A mutant);

30

ACCEPTED MANUSCRIPT indicating the importance of the suggested conformational change in facilitating 30S binding: The conformational change likely exposes a surface that binds 30S. In the GDP:GTP state,

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while the Kd for 30S is 0.3 μM, that for 70S is 1μM – which is a difference of an order. Based on this, it may be suggested that, when 30S and 50S finally form 70S, EngA dissociates from

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the ribosome as a consequence of its poorer affinity towards 70S complex (Fig. 11). Overall, it appears that EngA acts as a checkpoint preventing the association of premature 50S (pre-

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SUPPLEMENTARY MATERIAL

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50S) with 30S. However, rigorous experimentation is required to establish this view.

Supplementary Data are available and includes the Supplementary Method, Supplementary

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Figures S1, S2, S3, S4, S5 and S6, and a video of SMD on YphC for the dissociation of GD1

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domain from GD2-KH domain.

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ACKNOWLEDGEMENTS

SM acknowledges CSIR for fellowship. AA and SKT acknowledge MHRD for fellowship. The authors acknowledge all BP lab members especially Varun Bhaskar and Prashant Kumar for initial analysis of interfaces and for their help in generating some of the mutants as well. We acknowledge Saravanan Murugeson for his assistance in the MD simulation studies. We also acknowledge Ministry of Human Resources and Development, India, for establishing a ‘Center of Excellence for Chemical Biology’ at IIT Kanpur. BP thanks Director, CSIRCFTRI, Mysore for his support and encouragement.

31

ACCEPTED MANUSCRIPT FUNDING

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This work was supported by grants from the Department of Biotechnology, India [grant no. BT/HRD/34/01/2009(V)], Department of Science and Technology, India [grant no.

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SR/SO/BB-43/2009] and Indian Council of Medical Research, India [grant no.

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5/8/9/86/2008-ECD-1].

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22 Laskowski RA, MacArthur MW, Moss DS & Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291.

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ACCEPTED MANUSCRIPT 23 Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE & Berendsen HJC (2005) GROMACS: Fast, flexible, and free. J. Comput. Chem. 26, 1701–1718.

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multistage sampling. Chem. Phys. Lett. 21, 297–300.

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24 Patey GN & Valleau JP (1973) The free energy of spheres with dipoles: Monte Carlo with

25 Torrie GM & Valleau JP (1977) Nonphysical sampling distributions in Monte Carlo free-

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energy estimation - Umbrella sampling. J. Comput. Phys. 23, 187–199.

26 Torrie GM & Valleau JP (1974) Monte Carlo free energy estimates using non-Boltzmann

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sampling: Application to the sub-critical Lennard-Jones fluid. Chem. Phys. Lett. 28, 578–581.

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27 Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW & Klein ML (1983) Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935.

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28 Lindorff-Larsen K, Piana S, Palmo K, Maragakis P, Klepeis JL, Dror RO & Shaw DE

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(2010) Improved side-chain torsion potentials for the Amber ff99SB protein force field.

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Proteins 78, 1950–1958.

29 Berendsen HJC, Postma JPM, Gunsteren WF van, DiNola A & Haak JR (1984) Molecular dynamics with coupling to an external bath. J. Chem. Phys. 81, 3684–3690.

30 Hoover null (1985) Canonical dynamics: Equilibrium phase-space distributions. Phys. Rev. A 31, 1695–1697.

31 Nosé S (2002) A molecular dynamics method for simulations in the canonical ensemble. Mol. Phys. 100, 191–198.

32 Parrinello M & Rahman A (1981) Polymorphic transitions in single crystals: A new molecular dynamics method. J. Appl. Phys. 52, 7182–7190. 36

ACCEPTED MANUSCRIPT 33 Nosé S & Klein ML (1983) Constant pressure molecular dynamics for molecular systems. Mol. Phys. 50, 1055–1076.

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34 Izrailev S, Stepaniants S, Isralewitz B, Kosztin D, Lu H, Molnar F, Wriggers W &

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Schulten K (1999) Steered Molecular Dynamics. In Computational Molecular Dynamics: Challenges, Methods, Ideas (Deuflhard P, Hermans J, Leimkuhler B, Mark AE, Reich S, & Skeel RD, eds), pp. 39–65. Springer Berlin Heidelberg.

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35 Lemkul JA & Bevan DR (2010) Assessing the Stability of Alzheimer’s Amyloid

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Protofibrils Using Molecular Dynamics. J. Phys. Chem. B 114, 1652–1660.

36 Kumar S, Rosenberg JM, Bouzida D, Swendsen RH & Kollman PA (1992) THE weighted

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histogram analysis method for free-energy calculations on biomolecules. I. The method. J.

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Comput. Chem. 13, 1011–1021.

37 Recht MI, Ryder SP & Williamson JR (2008) Monitoring assembly of ribonucleoprotein

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complexes by isothermal titration calorimetry. Methods Mol. Biol. Clifton NJ 488, 117–127.

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38 Wiseman T, Williston S, Brandts JF & Lin LN (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179, 131– 137.

39 Shan S & Walter P (2003) Induced nucleotide specificity in a GTPase. Proc. Natl. Acad. Sci. U. S. A. 100, 4480–4485.

40 Naganathan A & Moore SD (2013) Crippling the essential GTPase Der causes dependence on ribosomal protein L9. J. Bacteriol. 195, 3682–3691.

37

ACCEPTED MANUSCRIPT Figure Legends

1.

A

structural

analysis

of

EngA[GTP:GDP],

EngA[GDP:GDP]

and

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Figure

EngA[GMPPNP:GMPPNP] states represented by Der, YphC and EngA respectively.

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(A). Structural superposition of EngA homologues, Der from T. maritima (TmDer PDB:1MKY, golden yellow) and YphC from B. subtilis (PDB:2HJG, purple) with a backbone rmsd of 0.984 Å for the GD2 and KH domains of the respective proteins. In both of

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these structures, GD2 is bound to GDP. Whereas GD1 of YphC is bound to GDP, in Der

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GD1 mimics the GTP bound state (see text, section 1); the two phosphates (indicated as PO4) that occupy positions equivalent to Pβ and Pγ phosphates of GTP at the nucleotide binding

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pocket are shown. Therefore, YphC and Der represent two distinct states EngA[GDP:GDP] and EngA[GTP:GDP], respectively. Structural comparison of these two shows that GD1

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undergoes a large movement of ~60Å between the GTP and GDP bound sates, whereas positions of GD2 and the KH domain remains largely unaltered. This movement results in the

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formation of two distinct GD1-KH interfaces, termed GD1GDP-KH and GD1GTP-KH, depicted

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by red and green broken lines, respectively. (B). Structure of E.coli EngA when in complex with 50S ribosomal subunit in the GMPPNP:GMPPNP state. It acquires an extended conformation (‘open state’) wherein GD1 no longer forms an interface with the KH domain, but the positions of GD2 and KH domain remains largely unaltered compared to the other structures. (C). Structural superposition of EngA homologues, from E.coli (PDB:3J8G, chain X, brown), YphC from B. subtilis (PDB:2HJG, purple) and Der from T. maritima (TmDer PDB:1MKY, golden yellow) with a backbone rmsd of 1.069 Å for the GD2 and KH domains of the respective proteins. Both G-domains of YphC is bound to GDP while in E.coli EngA is bound to GMPPNP and in Der, GD1 mimics the GTP bound state (see text, section 1) whereas GD2 is bound to GDP. Comparison of the structures reveal that YphC and Der GD1

38

ACCEPTED MANUSCRIPT occurs in a distinct interface (red and green lines respectively) with the KH domain, while in

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E.coli EngA the two domains are wide apart.

Figure 2. Conserved motifs at GD1GDP-KH interface. (A) A representative multiple

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sequence alignment of various EngA homologues is shown. The region spanning the GD1GDP-KH interface are shown. The secondary structure elements represented by cylinders for α-helices, arrows for β strands and lines for loop regions are shown. Conserved motifs

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labelled M1-[(E/D)ΦΨXΦΩ], M2-[KΦXYΩTQ87], M3-[ΦΦΦ(F/H)(V/G)N88] are indicated

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by red lines at the top of the alignment where Φ indicates the presence of a hydrophobic amino acid, Ψ indicates aromatic amino acids, Ω specifies the presence of small amino acids

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like Gly, Pro or Ala and X represents any amino acid. The number in subscript for a given residue indicates the percentage occurrence for that residue in the sequence alignment. EngA

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homologues from the following species are shown with the following abbreviations. EcEscherichia coli, Nm - Nitrococcus mobilis, Ba - Baumanniacic adellinicola, Af -

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Acidithiobacillus ferrooxidans, Nm2 - Neisseria meningitides, Vb - Verminephro

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bactereiseniae, Hm - Heliobacillus mobilis, St - Symbiobacterium thermophilum, Bs Bacillus subtilis, At - Anaerocellum thermophilum, Fp - Faecalibacterium prausnitzii, Da Desulfobacterium autotrophicum, Fb - Flavobacteriales bacterium, Tm -

Thermotoga

maritima, Pg - Phaeobacter gallaeciensis, Rp - Rhodopseudomonas palustris. The positions corresponding to the residues that were mutated and used in this study are shown in black circles in the respective motifs.

39

ACCEPTED MANUSCRIPT (B) The structure of YphC showing the GD1GDP-KH interface. Both GD1 (purple) and GD2 (olive green) is bound to GDP. GD1 (purple) forms an interface with KH (corn blue). The

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conserved motifs M1, M2 and M3 (golden yellow) at the GD1GDP-KH interface are depicted. The residues from the respective motifs that were mutated and used in this study are shown in

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red (also circled black).

Figure 3. Conserved motifs at GD1GTP-KH interface. (A) Similarly as in Fig.2, a

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representative sequence alignment of various EngA homologues with the secondary structure

sequence

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elements is presented for regions spanning the GD1GTP-KH interface. The conserved motifs,

M2-[KΦXYΩTQ87],

M3-[ΦΦΦ(F/H)(V/G)N88],

M4-

[(L/V)(V/I)DTG88GΦ], M5-[(M/V/I)XXQ81XXXA93I81], M6-[(R/K)I43P43T93ΩXL68N75] are

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indicated by red lines at the top of the alignment where Φ indicates the presence of a

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hydrophobic amino acid, Ψ indicates aromatic amino acids, Ω specifies the presence of small amino acids like Gly, Pro or Ala and X represents any amino acid. The number in subscript

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for a given residue indicates the percentage occurrence for that residue in the sequence

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alignment. The positons corresponding to the residues that were mutated and used in this study are shown in black circles in the respective motifs. EngA homologs are represented by the same abbreviations as in figure 2. (B) The structure of YphC (a homology model using Der crystal structure, PDB:1MKY as template) showing the GD1GTP-KH interface. GD1 (purple) is bound to two phosphates occupying positions of β and γ phosphates of GTP while GD2 (olive green) is bound to GDP. GD1 forms an interface with KH (corn blue). The conserved motifs M2, M3, M4, M5 and M6 (golden yellow) at the GD1 GTP-KH interface are depicted. The residues from the respective motifs that were mutated and used in this study are shown in red (also circled black).

40

ACCEPTED MANUSCRIPT Figure 4. Steered Molecular Dynamics of YphC (A) Force vs Time plot for the SMD simulation using a pull rate of 0.01 nm ps-1. Adjacent panels depict the trajectory snapshots at

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their respective time-points. The COM distance between the reference GD2-KH domain and GD1 domain at the relevant time-points are mentioned in square-brackets. (B) Residues of

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GD1-GD2 linker region (indicated by green dotted ellipse) involved in interactions (as red dotted lines) with both GD1 (in magenta) and GD2 (in yellow). (C) Interactions (as red dotted lines) that contribute to the stabilization of GD1GDP-KH interface. KH and GD1

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domain are depicted in yellow and magenta, respectively.

Figure 5. Interactions at GD1-KH interface. (A) A structural analysis of the contacts seen at

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GD1GDP-KH interface, contributed by residues of the conserved motifs (M1, M2 and M3 in golden yellow) is shown. The residues mutated in this study are highlighted in red and circled

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(black), whereas Y134(*) was mutated previously [6]. (B) The interactions seen at GD1GTP-

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KH interface contributed by residues of the conserved motifs (M2, M3, M4, M5 and M6 in golden yellow) are shown. The residues mutated in this study, are highlighted in red and

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circled (black). (C-G) Nucleotide binding to the YphC GD1-KH interface mutant proteins was assayed using fluorescent mant nucleotides, as described in ‘section 2.6’. The combinations of protein and nucleotide used are specified in the insets with equivalent colors and numbers. Emission spectra suggest that the mutants, (C) YphCD297N, (D) YphCY384AD297N,

(E)

YphCF398AD297N,

(F)

YphCN360AD297N

(G)

YphCI56AD297N are capable of binding to guanine or xanthine nucleotides and is unaffected by the mutations at GD1-KH interface.

41

ACCEPTED MANUSCRIPT

Figure 6. YphC proteins devoid of interface mutations fail to interact with the ribosome.

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The calorimetric titration of (A) YphC-WT in GMPPNP:GMPPNP state with 50S, (B) YphCD297N in GMPPNP:XMPPNP state with 50S, is shown. The upper panel represents the raw

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ITC data obtained upon 20 injections, 2μl each of 10μM of protein into 280μl of 1μM of ribosome. The heat change obtained with each injection was feeble, indicating no interaction

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of the protein with 50S ribosome.

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Figure 7. YphC-Y134A/D297N binds to the 30S, 50S and 70S ribosomal subunits in 1:1 stoichiometry. The calorimetric titration of (A) YphC-Y134A/D297N in GMPPNP:XMPPNP

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state with 50S, (B) YphC-Y134A/D297N in GDP:XMPPNP state with 50S, (C) YphCY134A/D297N in GDP:XMPPNP state with 30S, (D) YphC-Y134A/D297N in

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GDP:XMPPNP state with 70S is shown. The upper panel represents the raw ITC data

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obtained upon 15-20 injections, 2μl each of 10-15μM of protein into 280μl of 0.5-1μM of ribosome. The lower panel represents the integrated heats derived from the raw ITC data after

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subtracting the dilution enthalpy of the protein. The experiments were conducted at 300C as described in ‘Materials and Methods’ in duplicates. The lower panel also depicts the curve fitted using the ‘one-site-binding-model’.

42

ACCEPTED MANUSCRIPT Figure 8. YphC-Y384A/D297N binds to the 30S, 50S and 70S ribosomal subunits in 1:1 stoichiometry. The calorimetric titration of (A) YphC-Y384A/D297N in GMPPNP:XMPPNP

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state with 50S, (B) YphC-Y384A/D297N in GDP:XMPPNP state with 50S, (C) YphCY384A/D297N in GDP:XMPPNP state with 30S, (D) YphC-Y384A/D297N in

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GDP:XMPPNP state with 70S is shown. The upper panel represents the raw ITC data obtained upon 20 injections, 2μl each of 10-15μM of protein into 280μl of 0.5-1μM of ribosome. The lower panel represents the integrated heats derived from the raw ITC data after

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subtracting the dilution enthalpy of the protein. The experiments were conducted at 300C as

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described in ‘Materials and Methods’ in duplicates. The lower panel also depicts the curve

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fitted using the ‘one-site-binding-model’.

Figure 9. YphC-F398A/D297N binds to the 30S, 50S and 70S ribosomal subunits in 1:1

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stoichiometry. The calorimetric titration of (A) YphC-F398/D297N in GMPPNP:XMPPNP

F398A/D297N

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state with 50S, (B) YphC-F398A/D297N in GDP:XMPPNP state with 50S, (C) YphCin

GDP:XMPPNP

state

with

30S,

(D)

YphC-F398A/D297N

in

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GDP:XMPPNP state with 70S is shown. The upper panel represents the raw ITC data obtained upon 20 injections, 2μl each of 10-15μM of protein into 280μl of 0.5-1μM of ribosome. The lower panel represents the integrated heats derived from the raw ITC data after subtracting the dilution enthalpy of the protein. The experiments were conducted at 300C as described in ‘Materials and Methods’ in duplicates. The lower panel also depicts the curve fitted using the ‘one-site-binding-model’.

43

ACCEPTED MANUSCRIPT Figure 10. Disruption of GD1-KH interface is critical for EngA-50S interaction. (A). Structural superposition of GD1 domain of EngA homologues, Der from T. maritima (TmDer

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PDB:1MKY, golden yellow), YphC from B. subtilis (PDB:2HJG, purple) and E.coli EngA (PDB: 3J8G, chain X, brown) with a backbone rmsd of 1.268Å. YphC-GD1 is bound to

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GDP, TmDer-GD1 represents GTP mimic while E.coli EngA-GD1 is bound to GMPPNP. The inset focusses at the H-loop which lies between the β6 and α5 of GD1 and provides an invariant His residue for interaction with G2286 of the 23S rRNA (blue). E.coli H147 in

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contrast to TmDer H149 and YphC H148 occurs in a conformation suitable for interaction

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with the 23S rRNA. (B-D) Focusses at the GD1-KH interface of YphC (B) TmDer (C) and E.coli EngA (D). The conserved motifs at the interface, M1-M6 (golden yellow) are

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indicated. The M1 motif connects the H-loop (dark grey) and NKXD motif (dark grey). (E) Structural superposition of EngA homologues, from E.coli (PDB:3J8G, chain X, brown) and

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Der from T. maritima (TmDer PDB:1MKY, golden yellow) with a backbone rmsd of 1.096 Å for the GD2 and KH domains of the respective proteins. Both G-domains of E.coli EngA is

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bound to GMPPNP and in TmDer, GD1 mimics the GTP bound state (see text, section 1)

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whereas GD2 is bound to GDP. Structural comparison reveals that F414, the residue corresponding to Y437 of E.coli EngA is stabilized in a flipped position by a V372 from α12 of the TmDer-KH domain. α12 contains the M6 motif (dark grey) which interacts with M5 at α8 of GD1 (dark grey) to form the GD1GTP-KH interface in TmDer. In E.coli EngA either motifs, M6 from α12 of KH domain and M5 from α8 of GD1, (light grey) occur wide apart.

Figure 11. An overview suggesting the probable role of EngA in ribosome assembly.

44

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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Figure 9

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Figure 10

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Figure 11

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55

ACCEPTED MANUSCRIPT Table 1. Summary of EngA-ribosome interaction in different nucleotide bound states. EngA GD1:GD2

Experimental

Binding study

nucleotide state

Structure

results

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Remarks

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GD1[GTP] likely blocks

Cryo-EM bound to

Binds to 50S [on

GTP:GTP

(and 70S) [on

GD1[GDP] relieves the

interface disruption]

block on 30S binding.

GD2[GTP] is essential

Der T. maritima

Does not bind

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GTP:GDP

for binding to any ribosomal subunit

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GD2[GTP] is essential

YphC B.subtilis

Does not bind

for binding to any ribosomal subunit

AC

GDP:GDP

shown to bind 30S.

GD1[GTP] to

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NA

binding. KH domain

Binds to 50S, 30S

ED

GDP:GTP

somewhat aids in 50S

interface disruption]

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50S

interaction with 30S and

56

ACCEPTED MANUSCRIPT Table 2: Motifs at the interface of GD1/GD2 and KH domains. Consensus

Interface

Region

M1

[(E/D)ΦΨXΦΩ]

GD1GDP-KH

α4 of GD1

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Motif

[ΦΦΦ(F/H)(V/G)N88]

M2

SC RI

GD1GDP-KH and

β15 of KH

GD1GTP-KH

GD1GDP-KH and

[KΦXYΩTQ87]

β14 of KH

GD1GTP-KH

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M3

GD1GTP-KH

switch II of GD1

[(M/V/I)XXQ81XXXA93I81]

GD1GTP-KH

helix α8 of GD1

[(R/K)I43P43T93ΩXL68N75]

GD1GTP-KH

helix α12 of KH

[(L/V)(V/I)DTG88GΦ]

M5 M6

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M4

Here, Φ indicates the presence of a hydrophobic amino acid, Ψ indicates aromatic amino acids, Ω specifies the

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presence of small amino acids like Gly, Pro or Ala and X represents any amino acid. The number in subscript for a given residue indicates the percentage occurrence for that residue in the sequence alignment. For instance,

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Q87 indicates that Gln would be found 87 (out of 100) times, at that position in the alignment, while all those amino acids without a subscript implies that it is completely conserved.

57

ACCEPTED MANUSCRIPT Table 3. Determination of thermodynamic parameters for binding of YphC mutants (Y134A/D297N, Y384A/D297N and F398A/D297N) to the ribosomal subunits (50S, 30S

Ribosom

∆H

mutant

bound state

al

(kcal/mo

subunit

l)

50S

-969.9

GMPPNP:XMPP

7N

NP

Y134A/D29

GDP:XMPPNP

50S

GDP:XMPPNP

30S

PT

Y134A/D29

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7N

Kd

Stoichiomet

c-

(kcal/mol/de

(nM

ry (n)

valu

g)

)

-3.22

78.7

-984.9

7N

AC

Y134A/D29

GDP:XMPPNP

e 1.1 ± 0.189

±

14 ± 2.5

0.35 -3.27

100

1.06 ± 0.168

±

10 ± 2

8.5 -2240

-7.37

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7N

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Y134A/D29

∆S

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Nucleotide

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YphC

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and 70S) in presence of specified nucleotides (GDP/GMPPNP and XMPPNP) using ITC.

374.

0.452 ±

1.2

5±

0.036

±

23.8

0.02

5 70S

-714.5

-2.33

108

1.2 ± 0.00

8±

1± 0.05

18 Y384A/D29

GMPPNP:XMPP

7N

NP

50S

-963

-3.2

72.4

1.08 ± 0.19

±

15 ± 1.3

8.3 Y384A/D29

GDP:XMPPNP

50S

-1040

7N

-3.46

103. 9± 2.05

58

1.1 ± 0.24

11 ± 2.5

ACCEPTED MANUSCRIPT Y384A/D29

GDP:XMPPNP

30S

-605

-1.97

292

7N

0.96 ± 0.21

±

3± 0.00

38.7

GDP:XMPPNP

70S

-2060

7N

-6.8

163

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Y384A/D29

PT

5

0.859 ± 0.07

6±

1± 0.00

100

GMPPNP:XMPP

N

NP

GDP:XMPPNP

50S

30S

-924

GDP:XMPPNP

70S

183.

0.819 ± 0.03

8±

4± 0.5

4.05 -3.07

578

0.96 ± 0.1

±

2± 0.00

1.5 -1530

-1030

-5.10

-3.43

402

1.4 ±

8.75

0.26

719

22.5

59

1.15 ± 0.08

±

±

AC

F398A/D297

CE

N

N

-4.46

PT

GDP:XMPPNP

ED

N

F398A/D297

-1340

MA

F398A/D297

50S

NU

F398A/D297

0.95 ± 0.185

1± 0.00

Graphical abstract

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ACCEPTED MANUSCRIPT

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ACCEPTED MANUSCRIPT Highlights 1. GD1-KH interface is an important regulator of EngA-ribosome interactions.

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2. Large conformational change in GD1 due to GTP hydrolysis alters GD1-KH interface.

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3. Two distinct GD1-KH interfaces are stabilized by distinctly conserved sets of motifs.

61