Protein–protein interactions: a supra-structural phenomenon demanding trans-disciplinary biophysical approaches

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ScienceDirect Protein–protein interactions: a supra-structural phenomenon demanding trans-disciplinary biophysical approaches Olwyn Byron1 and Bente Vestergaard2 Responsive formation of protein:protein interaction (PPI) upon diverse stimuli is a fundament of cellular function. As a consequence, PPIs are complex, adaptive entities, and exist in structurally heterogeneous interplays defined by the energetic states of the free and complexed protomers. The biophysical and structural investigations of PPIs consequently demand hybrid approaches, implementing orthogonal methods and strategies for global data analysis. Currently, impressive developments in hardware and software within several methodologies define a new era for the biostructural community. Data can be obtained at increasing resolution, at relevant time-scales and under increasingly relevant experimental conditions, intricate data are interpreted reliably, and the questions posed and answered grow in complexity. With this review, highlights from the study of PPIs using a multitude of biophysical methods, are reported. The aim is to depict how the elucidation of the interplay of structures requires the interplay of methods. Addresses 1 School of Life Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK 2 Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark Corresponding authors: Byron, Olwyn ([email protected]) and Vestergaard, Bente ([email protected])

Current Opinion in Structural Biology 2015, 35:76–86 This review comes from a themed issue on Protein–protein interactions Edited by Ozlem Keskin and Alexandre Bonvin

http://dx.doi.org/10.1016/j.sbi.2015.09.003 0959-440/Published by Elsevier Ltd.

PPIs and the need for hybrid approaches Protein:protein interactions (PPIs) are dynamic entities and should be understood as a conformational and energetic multi-dimensional landscape formed in the interplay between multiple structural states of individual protomers, leading to transiently formed contacts and mixtures of stable and unstable structural configurations. This challenges us to study PPIs at different levels of resolution and Current Opinion in Structural Biology 2015, 35:76–86

on different timescales, and the ability to respond to the environment necessitates investigations under multiple experimental conditions. Here, we use the term suprastructure for a global consideration of the relevant multitude of structural levels. We opine that supra-structural investigations are necessary to obtain fundamental insight into the link from the statistical diversity of PPIs to macromolecular functionality. This calls for hybrid approaches, that is, the application of and simultaneous data analysis from orthogonal biostructural and biophysical methods, some of which are summarised in Table 1 and illustrated in Figure 1. In this article we describe prime recent examples of the elegant analysis of complex PPI behaviour, with emphasis on hybrid structural studies.

Assessment of binding affinity The affinity between protomers drives complex formation. The binding free energy can be determined from various methods (Figure 2), including surface plasmon resonance (SPR), microscale thermophoresis (MST), isothermal titration calorimetry (ITC) and analytical ultracentrifugation (AUC). These methods can be label-free, but particularly strong binding can be monitored using fluorescently labelled proteins and AUC. While sensitivity is advantageous, labelling can potentially alter the results. As an example, in a study of the self-association of the amino-terminal domain (ATD) of GluA2 [4] it was demonstrated that the life-time of the 5,6-carboxyfluorescein-GluA2 ATD dimer (which has a Kd of 2.3 nM) is significantly longer than that of the Dylight488(Kd = 20.5 mM) or EGFP- (Kd = 25.4 nM) labelled forms. SPR was applied in a study aimed at developing more efficient anti-Plasmodium falciparum (Pf) vaccines. Regions of PfEBA175 were investigated for their complex formation with the erythrocyte surface protein GYPA [55]. Fitting to SPR sensorgrams of varying contact times identified a bivalent mode of interaction and determined PfEBA175 regions facilitating GYPA interaction by promoting PfEBA175 dimerisation. MST is most commonly used for the determination of Kd [34] but is advantageous over some other methods in allowing measurement against complex backgrounds, for example, in cell lysates. Seidel et al. [35] measured Kd for the interaction between TEM-1 b-lactamase and its inhibitor protein BLIP, and revealed a correlation between conformational changes and the trapping of waters at the BLIP-TEM-1 interface. The comparison between buffer and cell lysate conditions surprisingly revealed the opposite of the generally www.sciencedirect.com

Structural complexity of protein:protein interactions Byron and Vestergaard 77

Table 1 Summary of some of the key biophysical methods that can be used to study PPIs. Brief description

What can be determined

Range of applicability

AFM

Atomic force microscopy is a type of scanning probe microscopy in which the surface roughness of a sample in solution is mapped at a resolution > 1000 times that imposed by the optical diffraction limit.

3D surface image; mechanical properties of surface.

Sample environment can be ambient air or liquid, including in vivo. Limited scan image size (150 mm  150 mm) and depth of field (20 mm).

[1]

AUC

Analytical ultracentrifugation measures the time-dependent change in, or equilibrium, concentration distribution of macromolecules in solution under a centrifugal field.

Sedimentation coefficient (s (S)), translational diffusion coefficient (Dt (cm2 s 1)), mass (M (g mol 1)), second virial coefficient (ml mol g 2), Kd (M), stoichiometry.

M: 100–5  106 g mol 1. s: 0.1–100 S. Kd: pM–mM.

[2,3,4–10]

cryo-EM

Cryo-electron microscopy is a form of transmission electron microscopy that images samples cooled to 77 K with a resultant increase in image resolution compared with conventional TEM.

High-resolution images of samples. Tomographic reconstruction possible.

Resolutions of  2 A˚ have recently been achieved. Samples do not have to be stained or crystallised. Relatively large particles are still required, and complexes must be stable at dilute conditions.

[1,7, 11–18,19]

DLS

Dynamic light scattering, also known as photon correlation spectroscopy or quasi-elastic light scattering, measures fluctuations in scattered light intensity due to proteins diffusing in and out of laser light path.

Translational diffusion coefficient (Dt (cm2 s 1)), hydrodynamic radius (Rh (nm)) if sample is monodisperse, particle size distribution.

Rh: 0.3 nm–10 mm. Dt: 7  10 6– 2  10 10 cm2 s 1.

[2,3,8,20]

FA

Fluorescence anisotropy can be used to measure the binding constants and kinetics of reactions that cause a measurable change in the rotational lifetime of the molecules, provided at least one of the binding partners is fluorescently labelled.

Anisotropy, polarisation, rotational correlation time (tcor (ns)).

Depolarised fluorescence can be used to analyse most PPIs. Gives a direct, near instantaneous measure of bound/free label ratio and hence equilibrium binding constant.

[10]

FCS

Fluorescence correlation spectroscopy is the fluorescent analogue of DLS and offers the possibility of high sensitivity in the observation of PPIs through the change in Dt by the use of fluorescently labelled binding partners.

Translational diffusion coefficient (Dt (cm2 s 1)), hydrodynamic radius (Rh (nm)).

[fluorophore]: pM–nM. Volumes as low as fl (mm3).

[21]

FRET

In Fo¨rster resonance energy transfer the interaction between two fluorescentlylabelled molecules (termed donor and acceptor) is detected by monitoring changes in the fluorescence emitted by the donor or acceptor. This is an ensemble method.

Whether or not two molecules are interacting, that is, the donor and acceptor are closer than the FRET distance.

FRET distance: 1–10 nm.

[4,22,23]

HDX-MS

In hydrogen-deuterium exchange-mass spectrometry the solvent accessibility of a molecule is determined by mass spectrometry of its proteolytic fragments.

Location and relative amount of H/D exchange along the peptide backbone.

Single amide resolution is achievable.

[24–26]

IMS

Ion-mobility mass spectrometry uses frictional encounters with inert gas molecules to separate macromolecular ions on the basis of not only their charge but also shape.

Rotationally averaged collision cross section (CCS (A˚2)).

Resolution in CCS of <3 A˚2 has been reported.

[27]

Technique

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References in this review

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78 Protein–protein interactions

Table 1 (Continued ) Technique

Brief description

What can be determined

Range of applicability

References in this review

ITC

Isothermal titration calorimetry is a labelfree calorimetric study of molecular interactions that directly observes the change in heat of a solution associated with a change of composition during titration of a macromolecular component with increasing amounts of its binding partner(s).

DQ (J mol 1), Kd (M), molar ratio, DH (J mol 1), DS (J K 1 mol 1), cooperativity

Kd: pM–mM.

[3,5,8,10, 28,29]

MS

Mass spectrometry is an umbrella term describing numerous methods for determining the amount and chemical nature of components in a system in terms of their mass-to-charge ratio (m/z).

m/z (unitless) as a function of intensity (and possibly time).

Mass of system: ions — viruses.

[30–33]

MST

Microscale thermophoresis follows the thermophoresis-dependent depletion or accumulation of fluorescently-labelled molecules within a temperature gradient.

Kd (M), stoichiometry, DH (J mol 1) DS (J K 1 mol 1).

Mass of system: ions — ribosomes. [fluorophore]: pM– nM. Kd: 10 pM–mM (with extrinsic labelling) or 10 nM–mM with intrinsic fluorescence.

[3,34, 35,36]

NSE

Neutron spin echo spectroscopy records the inelastic broadening of SANS (see below) intensity and thereby provides information on nanoscale macromolecular motion.

I(Q,t) as a function of t from which the effective diffusion coefficient as a function of Q (Deff(Q)) can be determined.

I(Q,t): nm-sub-mm. t: ns–ms.

[37,38]

SANS

Small-angle neutron scattering is a noncrystalline diffraction method wherein neutrons are scattered elastically by macromolecules in solution to generate a scattering curve of intensity of scattered neutrons as a function of scattering angle (Q). Variations are elastic incoherent and quasi-elastic coherent NS.

Radius of gyration (Rg, nm), molar mass (g mol 1), distance distribution, stoichiometry, shape.

Mass range: 1 kDa — several hundred MDa.

[7,8,39, 40,41]

SAXS

Small-angle X-ray scattering is similar to SANS, but X-rays are used in place of neutrons. Variations are wide angle (WAXS) and time-resolved (TR) SAXS/ WAXS.

As for SANS.

As for SANS.

[7,8,29,38, 39,41–52]

SEC-MALLS

In size exclusion chromatography-multiangle laser light scattering the sample is separated on the basis of size before being characterised by MALLS.

Molar mass (g mol 1), radius of gyration (Rg (nm)).

Mass range > 200 kDa.

[20]

smFRET

Single molecule FRET is similar to FRET, except that a single donor and acceptor FRET pair is excited and detected.

Kd (M).

FRET distance: 1–10 nm.

[21,53,54]

SPR

In surface plasmon resonance the binding of molecules to a substrate (usually coated with a binding partner) is measured by detecting a change in the angle of reflection of the incident light.

kon (M

No lower molecular weight limit. [analyte]  10 pM. 3  109  kon  103 M 1 s 1. 1  koff  10 5 s 1.

[3,5,10, 55,56]

1

s 1), koff (s 1), Kd (M).

accepted trend for a decrease in Kd in crowded environments [57]. In an elegant example of its complementarity with other methods, MST was used by Xiong et al. [36] in combination with bio-layer interferometry and macromolecular crystallography (MX) establishing that avidity of the ferret-transmissible influenza (H5) virus is proportional to (Kd)m where m is the number of interactions between the viral receptor and hyaluronic acid. This means that even a modest decrease in Kd can lead to orders of magnitude drop in viral avidity. Current Opinion in Structural Biology 2015, 35:76–86

While PPI is normally understood as the combination of functionally relevant interactions, also unwanted PPI (such as aggregation in biopharmaceutical preparations) are relevant to study and control. Saito et al. [2] conducted a rigorous biophysical assessment of the impact of ionic strength and sugars on the propensity for aggregation of monoclonal antibodies (MAb) using sedimentation equilibrium (SE) AUC, which reports on the degree of intermolecular interactions in relatively dilute solutions and can be used to assess colloidal stability. AUC analysis, www.sciencedirect.com

Structural complexity of protein:protein interactions Byron and Vestergaard 79

Figure 1

data sets emanating from different biophysical methods comprising very different numbers of data, and thus being disproportionately weighted in the global analysis, by choosing a weighting factor related to the square root of the number of data (rather than the number of data directly). In so doing, they observed that none of the biophysical methods was, individually, able to generate well-determined binding energetics, providing instead unlimited error intervals for both binding constants and both binding enthalpy changes, whereas GMMA leads to well-defined enthalpy changes by breaking the crosscorrelation with affinity constants. Since errors in concentration determination or the presence of inactive fractions of material can lead to unphysical, non-integer numbers of binding sites, SEDPHAT uses concentration correction factors, and the user can include artificial data to evaluate which set of biophysical techniques and experimental conditions would increase the information content optimally [5]. Such hybrid analysis approaches are central to the analysis of supra-structural phenomena. Current Opinion in Structural Biology

Mixed oligomeric states and varying shapes The study of macromolecular interplays demands the interplay of biophysical methods. Macromolecular function is mediated via a multitude of energetically defined interactions. Interactions are relevant to study on different time-scales and length-scales, and at different levels of complexity. Hybrid methods, that is, global simultaneous analysis of data from orthogonal methods, increase the means to approach the intricate networks of the complex adaptive PPIs on an appropriate level. In this imaginative schematic of the diverse aspects of PPIs that can be investigated with hybrid biophysical methodology, two proteins are interacting: one is depicted as a purple ribbon with flexible N-termini and C-termini and one particularly flexible loop, the other as a dark orange ab initio SANS/SAXS bead model. Other colours are as follows: red — measurable parameters; yellow — surface topology elucidated by AFM or cryo-EM; yellow-green/blue sphere pair — FRET labels; green — collision cross section (CCS) elucidated by IMS; aqua blue — water; pink — surface backbone hydrogens exchanged by deuteriums. This diagram, the lists of parameters and methods are not intended to be exhaustive, rather simply illustrative.

however, truly gains in strength if coupled with orthogonal methodologies. With the global multi-method analysis (GMMA) extension of SEDPHAT (originally developed for AUC data analysis) Zhao and Schuck [3] enable global analysis of data from ITC, SPR, AUC, fluorescence anisotropy and other spectroscopic and thermodynamic methods (Figure 2). GMMA demonstrates that the whole is greater than the sum of its parts: global analysis gives statistically robust fitting and can resolve aspects of PPI that would otherwise be undetectable. For example, in GMMA analysis of SPR, ITC, sedimentation velocity (SV) AUC and fluorescence anisotropy (FA) data for the binding of a-chymotrypsin to two symmetric binding sites on soybean trypsin inhibitor, Zhao and Schuck [10] overcame the problem posed by www.sciencedirect.com

Protein complexes are often mixtures of stoichiometric states and their formation can include structural changes. Together, these factors challenge structural investigation. X-ray, neutron and light scattering are versatile techniques for the analysis of solution structure. As an example, in a study of the ligand binding domain of AMPA receptors, SAXS data reveal how allosteric modulators stabilise certain dimeric conformers, demonstrating cooperative structural changes in individual protomers, where the binding event could be simulated only if a four-body binding equilibrium model was used [46]. Even more complex binding models are necessary to describe amyloid fibrillation, a prime example of suprastructural complexity. A qualitative comparative structural study using primarily SAXS and electron spin resonance (ESR) spectroscopy revealed how bound metal pair-distances in different dimeric superoxide dismutase 1 (SOD1) mutants correlate with aggregation kinetics and clinical severity of amyotrophic lateral sclerosis [52]. Two other seemingly conflicting studies elucidate the complexity of Ab(1-42) fibrillation. Early oligomeric states formed during fibrillation have been investigated using either AUC [6] or light scattering coupled directly to a size-exclusion chromatography setup (SEC-MALLS), complemented with Fourier transform infrared spectroscopy and electron microscopy (EM) [20]. While Wolff et al. [6] reveal the existence of multi-hexamers, Nichols et al. [20] identified much larger pre-fibrillar species, in fact revealing that the overall dimensions and MW of the solution state of Ab(1-42) oligomers were even larger than that of the corresponding protofibrils. An interesting aspect in this study, however, is the fact that TEM images Current Opinion in Structural Biology 2015, 35:76–86

80 Protein–protein interactions

Figure 2

concentrations experimental geometry m

sedimentation equilibrium

incompetent fractions χsyr heat of dilution, b

osmotic pressure

noise b(r), β(t) experimental geometry m, b

isothermal titration calorimetry

sedimentation velocity

reaction heats

ΔH ΔΔH

V translational friction

Di

si

surface plasmon resonance capture / competition baseline offset b

KD, ΔG, ΔΔG

dynamic light scattering # coherence areas I(0)

spectroscopic signals

rotational friction

fk

εi, Δεk fluorescence signals

molar count rate

fluorescence anisotropy

εi, Δεk circular dichroism and other spectroscopy

steady-state fluorescence

Current Opinion in Structural Biology

Orthogonal methods reduce ambiguity in data interpretation. The formation of PPIs is driven by changes in Gibbs free energy (represented by red text at the central intersection), which can be probed directly or indirectly by a multitude of methods. The Venn diagram depicts biophysical methods yielding data (illustrated in the associated graphical panels) that can be analysed using the global multi-method analysis (GMMA) in the program SEDPHAT in order to improve the accuracy of the resulting binding parameters by utilising novel statistical tools. Each set has a method title (black text) and methods are described individually or in groups by additional blue text. Observables that can be interpreted in terms of, for example, the Kd of PPI, are in bold black while the bold red of enthalpic changes measured by ITC highlights the direct measurement of these binding parameters. In grey are method-specific technical and ‘nuisance’ parameters. In the GMMA process, all of the data sets are fit with the chosen model, wherein the local and ‘nuisance’ parameters are optimised, compared with the experimental data, and a global measure for the goodness of fit is calculated, which is then optimised by non-linear regression. Source: This figure is reproduced from [3] under the terms of the Creative Commons Attribution Licence.

from the same preparations reveal the presence of globular spheroids, rather than larger species. Indeed the authors comment on these conflicting observations, which again must reflect how the experimental conditions associated with different methodologies (e.g. total protein concentration, different surface characteristics, different volume:surface ratios, among others) influence the solution state of the amyloid species. Also, the authors mention that no intermediate species are observed by SEC-MALLS between the monomeric state and the protofibril state. This could reflect that a Current Opinion in Structural Biology 2015, 35:76–86

potential intermediate state, which may be present prior to SEC, will either dissociate into monomers or further associate into a protofibrillar state on the column. By contrast, during the sedimentation velocity centrifugation analysis in the complementary study by Wolff et al. [6], multimers of hexamers are identified. Probably, both studies reveal relevant amyloid properties but the comparison of studies underlines the necessity to consider how the choice of methods and sample preparations may influence the final outcome of supra-structural investigations. www.sciencedirect.com

Structural complexity of protein:protein interactions Byron and Vestergaard 81

In two other studies, the high level of macroscopic heterogeneity of fibrils is investigated, focusing on peptide fragments of the amyloidogenic proteins transthyretin [1] or yeast prion protein [45]. The hierarchical fibril morphology demands the use of orthogonal methods, since no single method can cover the length scales covered by fibril structures, ranging from the atomic details of the peptide backbone interactions and dense sidechain packing, over the nm-scale protofibril association to the macroscopic morphological variations defined by the multitude of fibril:fibril stackings and heterogeneity of fibril lengths. Fitzpatrick et al. [1] combine magicangle spinning NMR, fibre diffraction (FD), cryo-EM, scanning transmission EM and atomic force microscopy (AFM) to this end, while Langkilde et al. [45] restrict themselves to three methods (SAXS, FD and EM) at the cost of resolution. The use of SAXS, however, provides the means to investigate which smaller species may be present during the fibrillation process [45] since SAXS data are additive and can be decomposed. More detailed ab initio shape analysis was applied in the study of the MnmE:MnmG complex that modifies tRNA wobble basepairs [29]. While the MnmE homodimer exists in equilibrium between open and closed states, the MnmG equilibrium is between the monomeric and dimeric form, and depending on the hydrolysis state of the nucleotide co-factor, the homodimers form two types of complexes including one or two MnmE dimers. Here, SEC-SAXS enabled the investigation of relatively unstable or short-lived complexes. In the work by Fislage et al. [29] partial high-resolution information was available, hence the protomers in the complex could be identified by shape. When this is not the case, more demanding neutron scattering experiments from partially deuterated protein complex samples can assist in identifying individual components. The initially monomeric and intrinsically disordered adhesion protein CD44 (cytoplasmic tail) forms a hetero-tetrameric complex with the adaptor protein ezrin in the presence of the lipid signalling molecule PIP2 [8], and SANS studies pinpointed both the overall conformation of the entire structure and that of individual components (including the lipid positions). Mass spectrometry (MS) can be used to determine the stoichiometry and topology of protein complexes isolated directly from cells, including intermediates in equilibrating systems [33] and membrane protein complexes [30,31,58]. Advances in instrumentation and sample preparation [32] are permitting a new wave of progress in the study of intact membrane PPIs [31]. In ion-mobility MS (IMS) frictional encounters with inert gas molecules separate the macromolecular ions on the basis of not only their charge but also shape. IMS is faster and uses less sample than many other biophysical methods (e.g. SAXS, AUC or DLS) but it appears limited by assumptions made www.sciencedirect.com

concerning protein surface solvation [27]. It may be that a more sophisticated, protein-specific representation of hydration (reviewed by Rocco and Byron [59]) could rescale the collision cross section in a more objective manner.

A revolution in PPI investigation For decades, cryo-EM has played a significant role in establishing the overall structure of very large protein complexes. The information content in cryo-EM reconstructions has virtually exploded with the recent development of ultra-fast detectors (see the review by Nogales and Scheres [17]) and the corresponding development in image processing methodology [18,60]. The method now enables high-resolution reconstructions, notably from significantly fewer particles. This means that we can uncover details of PPI interfaces for very large complexes. The structural biology community has seen only the beginning of what is surely a new era, with recent splendid examples of high-resolution reconstructions of mitochondrial and bacterial ribosomes [11,15] or the protective antigen pore at 2.9 A˚ resolution [16]. Examples of the analysis of smaller complexes (<1 MDa), include the recent proteasome structure at 2.8 A˚ resolution [13] or b-galactosidase with a cell-permeant inhibitor at a stunning resolution of 2.2 A˚ [12]. New technological developments pave the way for analysis of increasingly smaller complexes. Also, with higher resolution and improved image processing software [18], it is increasingly possible to identify a number of structural states in the micrographs, for example, in a recent study, where three different ‘frozen’ conformational states were identified in a time-resolved study of ribosome assembly [14]. Indeed, the hardware and software development in this field has initiated a true revolution in structural biology.

The synergy of high-resolution and lowresolution orthogonal structural methods Docking algorithms can be successfully coupled with SAXS data to study PPIs, significantly improving the performance of either method [41,43,44,49]. Interestingly, it was also shown that MS data were not adequate to guide the procedure [44]. In several cases over the last decade, the synergy between SAXS and NMR data in structure determination has been exploited, and the integration of scattering data with the powerful structure determination program XPLOR-NIH fully integrates data analysis and ensemble optimisation with sophisticated model building [39]. And finally, the combination of powerful de novo protein structure prediction algorithms with NMR and SAXS data has enabled the modelling of the dimer interface of Aha1 [48], hence alluding to the future potential of ab initio structure determination from joint data sources.

Allostery and dynamics Allostery plays an interesting role in PPIs. While the PPI interface is often observed to play a regulatory role in the Current Opinion in Structural Biology 2015, 35:76–86

82 Protein–protein interactions

Figure 3

pRb–E1A–CBP/p300

E1A–pRb

pR b

ac

xit ycle e ell c n/c tio yla et

Cel l cy cle re gu l

ion at

Ternary hub

Trans E1A a crip ce tio ty n al lati re o gu

on n lati E1A

E1A–CBP/p300 Current Opinion in Structural Biology

Graphical visualisation of the induction of multi-functionality via suprastructural complexity. The intrinsically disordered protein E1A is a central hub for functional PPIs. E1A engages in complexes with several other proteins. Of these, complex formation with a few other regulatory proteins greatly influences the recruitment of other binding partners, as well as the general functional outcome of the complex formation. This initial complex state is defined as primary (grey), binary (blue; complex with the retinoblastoma protein pRb), ternary (green; complex with the general transcriptional co-activator CREB binding protein CBP and the CBP paralogue p300) or quaternary (red; complex state with all three coregulators). Each of these states is a second-level functional hub, specifically interacting with several protein partners (examples marked with a solid dot in the diagram). Several dots on concentric circles reveal PPIs with the same protein partner, and exemplify how the initial complex state (primary/binary/ternary/quaternary) results in different functional pathways. The figure reveals how functionality multiplies with promiscuity. In general, supra-structural diversity is achieved via multi-dimensional energy landscapes, enabling rapid cellular response to environmental changes. This figure is modified from [53] with permission.

activity of distant binding sites in protein complexes [46], it is increasingly evident that allostery also influences complex dynamics, and with functional consequences (Figure 3 and [53]). The homo-tetrameric 3-deoxy-Darabino-heptulosonate-7-phosphate synthase (DAH7P) binds various allosteric inhibitors in the PPI interface. High-resolution MX structures of the apo form and different ligand-bound forms of DAH7P revealed that the individual protomers did not change their overall conformation upon ligand binding. ITC, on the other hand, revealed how different ligand binding sites synergistically communicated, in accordance with MD simulations, suggesting very different dynamic effects of single-/dualligand binding, and hence in concert, MX, ITC and MD simulations show how the allosteric effect on enzymatic activity is caused by altered tetramer dynamics [28]. Interestingly, when the DAH7P tetramer forms a heterooctameric complex with chorismate mutase (CM), not only is the enzymatic activity of both enzymes drastically increased, but the allosteric effect residing on DAH7P is transferred to CM [9]. In all of these examples, the allosteric effect is mediated via PPI interfaces, while in other examples allostery influences the ability of proteins to undertake homo-complex or hetero-complex formation Current Opinion in Structural Biology 2015, 35:76–86

[61]. Indeed protein complexes are highly dynamic entities, as for example, evidenced by the NMR-based and SAXS-based evaluation of the HIV-capsid dimer ensemble structure [42]. Intrinsic disorder may even be a prerequisite for complex formation [50,53]. Particularly intriguing are the multi-site interactions between the nine substrate sites on the intrinsically disordered region of the cyclin-dependent kinase inhibitor Sic1 and the one active site of ubiquitin ligase CDC4. Indeed such complexes must be studied in solution, by for example, NMR [56] or single-molecule fluorescence [21]. Hydrogen/deuterium exchange (HDX) is a widely used, elegant and versatile approach which can be combined with MS, NMR or neutron crystal diffraction for the determination of residues involved in the formation of specific PPI interfaces. Whilst the spatial resolution of HDX-MS is lower than that of MX or NMR alone, it offers an advantage over HDX-NMR in that it is able to report on conformational dynamics and interface composition without an effective upper size limit (whereas that of HDX-NMR is 25 kDa) [26]. Shukla et al. [24] combined HDX-MS and single-particle negative-stain EM to characterise the b2V2R/b-arrestin-1 PPI. Both the www.sciencedirect.com

Structural complexity of protein:protein interactions Byron and Vestergaard 83

b2V2R/b — arrestin-1 interface and regions of elevated dynamics in b-arrestin-1 were identified and the data permitted constrained modelling of the constituent crystal structures within EM density resulting in a full model for the complex. Much of the giant multi-enzyme pyruvate dehydrogenase complex E2
E3BP core remains unmapped by MX owing both to its complexity (60 subunits) and extended, flexibly linked constituent subdomains (see e.g. [7]). Despite this, Wang et al. [25] have combined HDX-MS with NMR to define the interaction loci between the E2
E3BP core (M > 3 MDa) and two regulatory enzymes, pyruvate dehydrogenase kinases PDK1 and PDK2 (monomer mass  45 kDa). Excited, short-lived states are key to PPI formation. Rennella et al. [47] used primarily SAXS and NMR to show how homo-dimeric and hetero-dimeric states of the folding intermediate (I) and native (N) form of beta-2microglobulin exist in equilibrium including also the hetero-dimeric I:N form. Importantly, the I state has an increased aggregation propensity, eluding to the possibility that an excited state formed during dimerisation triggers the onset of amyloid formation. An elegant, recent study suggests that further aggregation of amyloid species may be driven by the energy associated with the formation of a high-entropy water layer forming on the surface of the fibrils [40]. Here, elastic incoherent and quasi-elastic neutron scattering were essential to visualise the water dynamics. Indeed, neutron-based methods and neutron spin echo (NSE) in particular seem suited to monitor protein dynamics on appropriate length-scales and time-scales (see Callaway and Bu [37] for a review) as examplified by another study, where the coupling between the solvent effect on insulin hexameric stability and amyloid aggregation propensity was investigated by the application of NSE, SAXS and fluorescence spectroscopy [38].

Frontiers and challenges: faster, smaller and more real Hybrid methods, a theme of this review, bridge between resolution limits of various methodologies and reduce the ambiguity of analysis, and this trans-disciplinarity will continue to grow. But what are the frontiers and challenges for biophysical studies of PPIs? The field is vividly developing, resulting in an exciting, kaleidoscopic horizon. Methodological development is taking biophysics into faster, smaller and more life-like realms: time-resolved direct observations of binding events at the single molecule level in living cells, the ability to embrace the heterogeneity of molecular behaviour arising from instrinsic disorder, flexibility and other dynamics, a far move away from the predominant current view of PPIs, which to a great extend is shaped by mainly static in crystallo structures of protein complexes. www.sciencedirect.com

The ability to follow macromolecular complexes in action at high resolution and in real-time emerges with timeresolved serial femtosecond crystallography (TR-SFX) at X-ray free electron lasers [62] or using high-brilliance synchrotron radiation [63]. The ultra-high intensities mean that micro-crystals and nano-crystals can be used, and the short pulses mean that snapshots can be captured with ultra-high time-resolution following different structural states upon inducing changes e.g. by laser or ultrafast mixing, both of which benefit from nano-sized crystals. Importantly, the setup enables the study of transient complexes and other unstable or sensitive systems. Successful examples include de novo structure determination from the photosystem II complex [64] and photoactive yellow protein [65]. Time-resolved solution scattering studies (TR-W/SAXS) avoid potential crystal lattice induced structural restrictions on macromolecular movement, but necessitate high-resolution prior information for adequate analysis. The potential of the method is well elucidated, in the fitting of structural changes of WT and mutant haemoglobin at 100 ns resolution to a two-state kinetic model, and the data support cooperative allostery in the tetrameric interface. Indeed structural changes are observable immediately upon the 3 ns photoinduction [51]. It is somewhat ironic to talk of single molecule approaches in a review concerned with PPIs, however, single molecule Fo¨rster resonance energy transfer spectroscopy (smFRET) directly detects the kinetics of individual binding events, building into histograms that describe the fractional populations of each state and thence the Kd of PPIs. smFRET is ideal for high-affinity and aggregation-prone systems, such as the E1A–CBP–pRb allosteric complex investigated by Ferreon et al. [53], as it uses low concentrations (100 pM) of fluorescently labelled protein and permits observation of the myriad PPIs in which promiscuous intrinsically disordered proteins (such as E1A) engage (Figure 3). A different example of the elegant analysis enabled by smFRET, is the study of time-dependent aggregation of low concentration tau amyloid revealing the energy landscape of the fibrillation process and the effect on this of pathological mutations [54]. The majority of structural and biophysical investigations are made in vitro, which in general improves the signal:noise ratio in data and provides well-defined milieus and optional single-parameter effect studies. The fundamental challenge of inferring in vivo behaviour from in vitro studies however remains significant, and it is exciting to follow current developments towards in-cell analysis. Detert Oude Weme et al. [22] have provided the first account of frequency domain-fluorescence lifetime imaging microscopy (FD-FLIM) to analyse FRET in single bacterial cells, wherein inter-individual fluorescence lifetimes can be resolved within a population of Bacillus subtilis. The multicolour TR-FRET microscopy study Current Opinion in Structural Biology 2015, 35:76–86

84 Protein–protein interactions

of GPCR oligomerisation and subsequent internationalisation demonstrates the potential of this system for the study of heterogeneity and multicomponent interactions in PPIs [23]. As was discussed above for cryo-EM, improved detector technology also revolutionises electron cryo-tomography (cryo-ET), and direct imaging of cellular or subcellular surfaces and their components is already possible, for example, the study of import super-complexes in the mitochondrial membrane [19]. The potential of imaging using XFELs can only yet be imagined, as suggested by the impressive recent imaging of live cyanobacteria, revealing data extending to nm resolution from highly complex macroscopic – notably live – particles [66]. With an increased resolution and improved methodologies, direct time-resolved imaging of in-cell macromolecular networks in action seems almost within reach.

Conclusions The high level of supra-structural complexity that defines macromolecular function necessitates hybrid methods in the study of PPIs. While intra-disciplinarity is a prerequisite, global data analysis from orthogonal methods greatly boosts the analysis potential, reducing ambiguity while fundamentally increasing resolution. Current evolution in methodology has created opportunities that have yet to be fully exploited within direct imaging, timeresolved analysis and various levels of in vivo investigations. All in all, current biophysical and structural investigations provide the first glimpse of as yet unexploited supra-structural phenomena, promising a conceptual revolution of our understanding of macromolecules when entering the era of Biostructure_2.0.

a cross-beta amyloid fibril. Proc Natl Acad Sci U S A 2013, 110:5468-5473. Saito S, Hasegawa J, Kobayashi N, Tomitsuka T, Uchiyama S, Fukui K: Effects of ionic strength and sugars on the aggregation propensity of monoclonal antibodies: influence of colloidal and conformational stabilities. Pharma Res 2013, 30:1263-1280. Sedimentation equilibrium (SE) in the analytical ultracentrifuge (AUC) was used to determine the second virial coefficient (B2) which reports on the degree of intermolecular interactions in relatively dilute solutions and can be used to assess colloidal stability. In this case, the MAb solution is considered to be a colloid. The sign (i.e. positive or negative) of B2 implies repulsive or attractive interactions, respectively and knowledge of the protein electrostatic surface can be used to interpret B2 in terms of the interaction of the protein with itself and with its solvent environment and hence guide strategies for aggregation control and the desired enhancement of colloidal stability.

2. 

3. 

Zhao H, Schuck P: Combining biophysical methods for the analysis of protein complex stoichiometry and affinity in SEDPHAT. Acta Crystallogr D-Biol Crystallogr 2015, 71:3-14. As part of a duo of complementary papers (cf Zhao, Piszczek & Schuck) documenting the development of a global multi-method approach (GMMA) to combining biophysical methods for the analysis of protein complex stoichiometry and affinity, this study describes the use of the program SEDPHAT for the global analysis of ITC, SPR, AUC, fluorescence anisotropy and other spectroscopic and thermodynamic data. The important take-home message is that the whole is greater than the sum of its parts: global analysis gives statistically robust fitting and can resolve aspects of PPI that would otherwise be undetectable. 4.

Zhao H, Lomash S, Glasser C, Mayer ML, Schuck P: Analysis of high affinity self-association by fluorescence optical sedimentation velocity analytical ultracentrifugation of labeled proteins: opportunities and limitations. PLoS ONE 2013, 8:e83439.

5.

Zhao H, Piszczek G, Schuck P: SEDPHAT — a platform for global ITC analysis and global multi-method analysis of molecular interactions. Methods 2015, 76:137-148.

6.

Wolff M, Unuchek D, Zhang B, Gordeliy V, Willbold D, NagelSteger L: Amyloid beta oligomeric species present in the lag phase of amyloid formation. PLoS ONE 2015, 10:e0127865.

7.

Vijayakrishnan S, Kelly SM, Gilbert RJC, Callow P, Bhella D, Forsyth T, Lindsay JG, Byron O: Solution structure and characterisation of the human pyruvate dehydrogenase complex core assembly. J Mol Biol 2010, 399: 71-93.

8.

Chen X, Khajeh JA, Ju JH, Gupta YK, Stanley CB, Do C, Heller WT, Aggarwal AK, Callaway DJ, Bu Z: Phosphatidylinositol 4,5bisphosphate clusters the cell adhesion molecule CD44 and assembles a specific CD44-ezrin heterocomplex, as revealed by small angle neutron scattering. J Biol Chem 2015, 290: 6639-6652.

9.

Blackmore N, Nazmi A, Hutton R, Webby M, Baker E, Jameson G, Parker E: Complex formation between two biosynthetic enzymes modifies the allosteric regulatory properties of both: an example of molecular symbiosis. J Biol Chem 2015, 290:18187-18198 1074/jbc.M115.638700:10.1074/ jbc.M1115.638700.

Conflict of interest statement Nothing declared.

Acknowledgements Many of the sub-headings in this review were inspired by an excellent talk delivered at the CEITEC Workshop ‘Current State-of-Art to Study Biomolecular Interactions and Assemblies in Life Sciences’ in November 2014 by Dr Patrick England (one of the coordinators of ARBRE https:// www.structuralbiology.eu/networks/association-resources-biophysicalresearch-europe). We are grateful to Dr Zhuo Li of NanoTemper Technologies GmbH for sharing with us her database of MST-related publications. BV is grateful for the inspiration during visits to the refuge ‘Klitga˚rden’, Skagen, Denmark, and both authors are grateful to the European Molecular Biology Organisation (EMBO) for the funding of Global Exchange Lecture Courses and Practical Courses that have provided the opportunity for discussion and collaboration on the topic of this review. BV acknowledges funding from the Danish Council for Independent Research, Sapere Aude program.

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