Solution Structure of the RIM1α PDZ Domain in Complex with an ELKS1b C-terminal Peptide

doi:10.1016/j.jmb.2005.07.047

J. Mol. Biol. (2005) 352, 455–466

Solution Structure of the RIM1a PDZ Domain in Complex with an ELKS1b C-terminal Peptide Jun Lu1, Hongmei Li2, Yun Wang2, Thomas C. Su¨dhof2 and Josep Rizo1* 1

Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd. Dallas, TX 75390, USA 2

Center for Basic Neuroscience Department of Molecular Genetics, and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd. Dallas, TX 75390, USA

PDZ domains are widespread protein modules that commonly recognize C-terminal sequences of target proteins and help to organize macromolecular signaling complexes. These sequences usually bind in an extended conformation to relatively shallow grooves formed between a b-strand and an a-helix in the corresponding PDZ domains. Because of this binding mode, many PDZ domains recognize primarily the C-terminal and the antepenultimate side-chains of the target protein, which commonly conform to motifs that have been categorized into different classes. However, an increasing number of PDZ domains have been found to exhibit unusual specificities. These include the PDZ domain of RIMs, which are large multidomain proteins that regulate neurotransmitter release and help to organize presynaptic active zones. The RIM PDZ domain binds to the C-terminal sequence of ELKS with a unique specificity that involves each of the four ELKS C-terminal residues. To elucidate the structural basis for this specificity, we have determined the 3D structure in solution of an RIM/ELKS C-terminal peptide complex using NMR spectroscopy. The structure shows that the RIM PDZ domain contains an unusually deep and narrow peptide-binding groove with an exquisite shape complementarity to the four ELKS C-terminal residues in their bound conformation. This groove is formed, in part, by a set of side-chains that is conserved selectively in RIM PDZ domains and that hence determines, at least in part, their unique specificity. q 2005 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: PDZ domain; RIM; active zone; protein NMR; binding specificity

Introduction PDZ domains are among the most abundant protein modules in metazoans.1–4 These modules of ca 100 residues function as protein–protein interaction domains, and often occur in modular proteins that serve as scaffolds to organize supramolecular signaling complexes. PDZ domains usually bind to short peptide motifs at the C terminus of target proteins.5,6 Binding commonly involves the C-terminal four or five residues of the peptide motif, and the side-chains at positions P0 and PK2 are very often the most critical for specificity (the position of the C-terminal residue is referred to as P0, and those of the preceding Abbreviations used: HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; ITC, isothermal titration calorimetry; TOCSY, total correlated spectroscopy. E-mail address of the corresponding author: [email protected]

residues as PK1, PK2, etc.). Thus, it was established early that many PDZ domains recognize C-terminal sequences with the motifs S/T-X-F (X, any amino acid; F, hydrophobic) or F-X-F, leading to their classification as class I and class II PDZ domains, respectively.1–4,7,8 The 3D structures of several PDZ domains with and without their bound ligands have shown that PDZ domains form compact folds with six b-strands (bA-bF) and two a-helices (aA-aB),9,10 and bind to their targets through a groove between strand bB and helix aB.9,11 The bound peptide forms a b-strand in an antiparallel orientation with respect to bB, with two side-chains (P0 and PK2) facing the PDZ domain groove and the other side-chains facing toward the solvent. In class I PDZ domains, a histidine residue forms a hydrogen bond with the hydroxyl group of the PK2 side-chain, whereas the histidine residue is replaced by a hydrophobic residue in class II PDZ domains, helping to form a hydrophobic cavity for the PK2 side-chain. Other common motifs that bind to PDZ domains

0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

456 such as X-X-C12,13 and D/E-X-F14 have been identified, leading to the definition of additional classes of PDZ domains. Moreover, some PDZ domains have unique binding specificities,15 others exhibit dual binding specificity,16 and some PDZ domains bind to internal sequences.17 Hence, PDZ domains are more versatile than was thought initially. Although the 3D structures of several of these less usual PDZ domains complexed with their ligands have been described,17–19 we are still far from understanding the full range of specificities existing within the PDZ domain family. An example of a PDZ domain with an unusual binding specificity is the PDZ domain found in RIMs.20 RIMs were identified initially as Rab3 effectors,21 and include four genes in mammals (RIM1, RIM2, RIM3g and RIM4g),22,23 and one in Caenorhabditis elegans (unc10).24 Mammalian RIM1 and RIM2 specify full-length transcripts encoding two closely related isoforms (RIM1a and RIM2a; referred to as a-RIMs) that contain an N-terminal zinc finger domain, a PDZ domain and two C-terminal C2-domains (referred to as C2A-domain and C2B-domain). The RIM2 gene also specifies a shorter transcript lacking the N-terminal zinc finger (RIM2b), and an even shorter transcript that, like RIM3g and RIM4g, encodes only for the C2B-domain and adjacent sequences.23 a-RIMs are large (ca 180 kDa) proteins localized at presynaptic active zones where neurotransmitters are released. Genetic experiments have shown that a-RIMs are critical for normal priming of synaptic vesicles to a release-ready state,24–26 and control some forms of short-term and long-term presynaptic plasticity.25–28 The severe impairment in learning and memory observed in RIM1a knockout mice emphasizes the importance of these multiple functions.29 The RIM PDZ domain binds to the C terminus of ELKS, a family of alternatively spliced proteins that are found at the active zone but exhibit a spectrum of subcellular distributions and expression patters.20 In addition, the other domains of RIMs have been reported to interact with multiple proteins, including Rab3s, which are small GTPases that regulate neurotransmitter release,30 Munc13-1,31 an active zone protein that is essential for synaptic vesicle priming,32–34 the Ca2C sensor synaptotagmin 1,25,35 the SNARE protein SNAP-25, which forms part of the membrane fusion machinery,35 the active zone proteins known as liprins,25 and RIM-BPs.22 These multiple interactions with proteins from the active zone and with components of the neurotransmitter release machinery suggest that RIMs form a protein scaffold that organizes the active zone and regulates neurotransmitter release. However, the structural basis for these interactions has not been characterized. The interaction between the RIM PDZ domain and ELKS may be of particular functional importance, since the PDZ domain is the most conserved among the RIM modules when the mammalian and C. elegans proteins are compared (58% sequence identity between rat RIM1a and unc10 PDZ domains24), and microinjection of a RIM1a fragment

Solution Structure of the RIM1a PDZ Domain

encompassing the PDZ domain impairs neurotransmitter release.36 In addition, the RIM PDZ domain/ELKS interaction exhibits an unusual specificity, since the four C-terminal residues of ELKS (GIWA) match approximately the profile of a class II PDZ-binding motif, but even conservative mutations in any of these four residues abolish binding.20 Interestingly, the RIM PDZ domain is closely related to the PDZ domain of piccolo, another large active zone protein that also binds to ELKS.36 However, as we describe below, the piccolo PDZ domain does not bind to the ELKS C-terminal sequence. Thus, the high level of specificity of the RIM PDZ domain/ELKS interaction may be critical for proper organization of the active zone. To understand the structural basis for this specificity, and to gain further insights into the structural features that determine the diverse specificities within the PDZ domain family, we have solved the 3D structure of the RIM1a PDZ domain in complex with an ELKS1b C-terminal peptide using NMR spectroscopy. The structure reveals that the four C-terminal residues from the peptide bind at an unusually deep and narrow groove of the RIM1a PDZ domain that contains well-defined binding pockets for each of these residues and that is lined, in part, by side-chains that are conserved selectively in RIM PDZ domains.

Results Specific binding of an ELKS1b C-terminal peptide to the RIM1a PDZ domain We first analyzed the binding of an 11 residue peptide (CDQDEEEGIWA) derived from the C-terminal sequence of ELKS1b (referred to as ELKS1bC) to the RIM1a PDZ domain using NMR spectroscopy. Unlabeled ELKS1bC peptide induced substantial shifts in a subset of cross-peaks of the 1 H–15N heteronuclear single quantum coherence (HSQC) spectrum of 15N-labeled RIM1a PDZ domain (Figure 1(a)), showing that the peptide binds to a specific region of the RIM1a PDZ domain. Conversely, addition of unlabeled RIM1a PDZ domain caused extensive shifts on the 1H–15N HSQC spectrum of the 15N-labeled ELKS1bC peptide (Figure 1(b)), also confirming the interaction. Analysis by isothermal titration calorimetry (ITC) (Figure 2) yielded a dissociation constant of 0.27 mM, which reflects a significantly higher affinity than those commonly observed for PDZ domain/target peptide interactions (1–10 mM dissociation constants).2 The high level of specificity of the RIM PDZ domain/ELKS interaction was demonstrated by extensive mutagenesis of the ELKS C-terminal sequence.20 To investigate this specificity further, we tested whether the ELKS1bC peptide binds to the piccolo PDZ domain, since it shares the highest level of sequence homology with the RIM PDZ domain among those in the databases, and piccolo

Solution Structure of the RIM1a PDZ Domain

457

Figure 1. Specific binding of the ELKS1bC peptide to the RIM1a PDZ domain analyzed by NMR spectroscopy. (a) H–15N HSQC spectrum of 15N-labeled RIM1a PDZ domain in the absence (black contours) and presence (red contours) of unlabeled ELKS1bC peptide. (b) 1H–15N HSQC spectrum of 15N-labeled ELKS1bC peptide in the absence (black contours) and presence (red contours) of unlabeled RIM1a PDZ domain. (c) 1H–15N HSQC spectrum of 15N-labeled ELKS1bC peptide in the absence (black contours) and presence (red contours) of unlabeled piccolo PDZ domain. 1

has been reported to interact with ELKS through a coiled-coil region.36 As shown in Figure 1(c), an unlabeled fragment containing the piccolo PDZ domain caused only very small perturbations on the 1H–15N HSQC spectrum of the 15N-labeled ELKS1bC peptide. Given the high concentration of the piccolo fragment used in these experiments (150 mM), this result shows that the ELKS1bC peptide has practically negligible affinity for the piccolo PDZ domain (at least 1000-fold lower than for the RIM1a PDZ domain). Three-dimensional structure of the RIM1a PDZ domain in complex with the ELKS1bC peptide To characterize the structural basis of the specificity of the RIM PDZ domain for the C-terminal sequence of ELKS, we determined the 3D structure in solution of the RIM1a PDZ domain complexed to the ELKS1bC peptide using NMR spectroscopy. Samples containing 15N-labeled or 15N,13C-labeled RIM1a PDZ domain and unlabeled ELKS1bC peptide, or vice versa, were used for resonance and nuclear Overhauser effect (NOE) assignments. To assign intermolecular NOEs, we used 2D 13Cfiltered,13C-edited NOE spectroscopy (NOESY)-HSQC spectra, as well as 3D 13C-edited NOESY-HSQC spectra with and without 13C-decoupling in the t1 dimension (Figure 3). In the latter spectra (Figure 3(a)), comparison of the cross-peak patterns observed with and without 13C-decoupling in the t1 dimension allows us to distinguish between intramolecular NOEs within the 13C-labeled chain (which split due to the 13C coupling in the

undecoupled spectrum) and intermolecular NOEs with the unlabeled chain (which are in the same position in both spectra). In our experience, these spectra yield substantially greater sensitivity than 3D 13C-filtered,13C-edited NOESY-HSQC spectra. Although the 3D 13C-edited NOESY-HSQC spectra with or without 13C decoupling in the t1 dimension generally yield higher resolution than 2D 13Cfiltered,13C-edited NOESY-HSQC spectra, the latter spectra facilitate assignment of NOEs close to the diagonal. Altogether, these data allowed us to assign 73 intermolecular NOEs to define the mode of binding of the ELKS1bC peptide to the RIM1a PDZ domain precisely. Final structures of the RIM1a PDZ domain/ ELKS1bC peptide complex were calculated using a total of 1568 restraints derived from the NMR data. A backbone superposition of the 20 structures with the lowest energies is shown in Figure 4(a), and a ribbon diagram of a representative structure is shown in Figure 4(b). The structural statistics are summarized in Table 1. As expected, the RIM1a PDZ domain forms a b-barrel structure with two b-sheets and two a-helices; one helix (aA) is short and is located at the top of the domain (in the orientation of Figure 4), while the other helix (aB) is longer and is located on the right side of the domain. However, the RIM1a PDZ domain contains seven b-strands, in contrast to most PDZ domains, which usually contain six b-strands. The additional b-strand in the RIM1a PDZ domain is formed by an N-terminal extension. We will refer to this strand as b0 and will use the common nomenclature for the six b-strands shared by most PDZ domains (bA-bF).

458

Solution Structure of the RIM1a PDZ Domain

RIM1a PDZ domain and other PDZ domains in the ˚ , but included a smaller PDB ranged from 2 to 3 A a number of equivalent C atoms. As an example, Figure 5(b) shows a superposition of the RIM1a PDZ domain with the third PDZ domain of PSD-95 complexed with a peptide ligand.9 These and other structure comparisons (not shown) revealed that the overall architecture of the RIM1a PDZ domain is analogous to those of other PDZ domains, with a similar core formed by the six shared b-strands and the two a-helices, but with marked differences in the loops connecting these elements of secondary structure. A distinctive feature of the RIM1a PDZ domain is that the N and C termini do not emerge at the same side of the domain, due to the presence of the additional, N-terminal b-strand (b0). This feature may be critical for the proper relative orientation of the sequences that precede and follow the RIM1a PDZ domain. It is worth noting that helix aB of the RIM1a PDZ domain is one turn longer than those of other PDZ domains (e.g. see Figure 5), which may be important for its unique specificity (see below). Structural basis of ELKS1bC peptide recognition by the RIM1a PDZ domain

Figure 2. Analysis of the affinity of the RIM1a PDZ domain/ELKS1bC peptide interaction by ITC. See Materials and Methods for details.

As is usually observed, the ELKS1bC peptide binds in an extended conformation between strand bB and helix aB of the RIM1a PDZ domain. The structure of the ELKS1bC peptide within the complex is well defined for the four C-terminal residues (Figure 4(a)), which account for most of the contacts with the RIM1a PDZ domain. The structure of the RIM1a PDZ domain is also generally well defined, but some heterogeneity is observed in a few loops, particularly in the loop connecting strands bA and bB. Part of this loop is characterized by sharp resonances and fast deuterium exchange rates, which indicate strongly the existence of flexibility. The overall quality of the structure of the RIM1a PDZ domain/ELKS1bC peptide complex is illustrated by the low deviations from the experimental restraints and from idealized covalent geometry, as well as by the Ramachandran map statistics (see Table 1). A search with DALI37 showed that, as expected, the RIM1a PDZ domain shares structural similarity with other PDZ domains. The highest Z score yielded by DALI (10.3) corresponds to the piccolo ˚ for PDZ domain, with a trace rms deviation of 2.7 A 95 equivalent Ca atoms (see superposition in Figure 5(a)). The trace rms deviations between the

The basis for the high level of specificity of the RIM PDZ domain becomes apparent upon examination of the mode of interaction between the ELKS1bC peptide and the RIM1a PDZ domain (Figures 6 and 7), and the sequence alignment shown in Figure 8. Overall, the binding mode conforms to the classical model of strand insertion between the strand bB and helix aB, and the C-terminal carboxyl group of the ELKS1bC peptide forms three hydrogen bonds with backbone NH groups of the RIM1a PDZ domain (from Leu630, Gly631 and Leu632). The peptide main chain forms three additional hydrogen bonds with strand bB. However, the surface representation of the RIM1a PDZ domain shown in Figure 6(a) reveals that the ELKS1bC peptide (in stick model) binds to an unusually narrow and deep groove of the RIM1a PDZ domain that is lined by bulky side-chains. Indeed, a survey of the structures of PDZ domain/ target peptide complexes available in the RCSB protein data base (PDB) showed that the peptidebinding grooves of PDZ domains are usually shallower than that of the RIM1a PDZ domain. This is illustrated by the surface representations of the third PDZ domain of PSD-95,9 the sixth PDZ domain of GRIP1,38 and the PDZ domain of nNOS,18 complexed to target peptides (Figure 6(b)-(d)); these correspond to class I, II and III PDZ domains, respectively. Note that, in general, a substantial portion of the side-chains in positions PK1 and PK3 of the peptide is highly exposed to the solvent, and that only the residue in position P0 inserts deeply into a pocket of the PDZ domain, although the nNOS PDZ domain also contains a deep pocket to accommodate the PK2 residue. In the RIM1a PDZ domain/ELKS1bC peptide complex,

Solution Structure of the RIM1a PDZ Domain

459

Figure 3. Analysis of intermolecular NOEs between the RIM1a PDZ domain and the ELKS1bC peptide. (a) Expansions of 3D 13Cedited NOESY-HSQC spectra acquired on a sample containing 15 N,13C-labeled ELKS1bC peptide and unlabeled RIM1a PDZ domain. Pairs of stripes taken at the 13C chemical shift of the IleK2 CG2 and IleK2 CD1 carbon atoms are shown. The upper and lower stripes in each pair correspond to spectra obtained without (Kdec) or with (Cdec) 13C decoupling in the t1 dimension. Intramolecular NOEs, which split in the upper stripes due to 13C couplings, are correlated with forked black lines. Intermolecular NOEs, which appear in the same positions in both spectra, are correlated with red lines. The crosspeak assignments are indicated. (b) Expansions of a 2D 13C-filtered, 13 C-edited NOESY-HSQC spectrum acquired on a sample containing 15N,13C-labeled RIM1a PDZ domain and unlabeled ELKS1bC peptide. Cross-peak assignments are indicated at the edges of the expansions.

the P0 residue from the peptide is inserted even more deeply into the PDZ domain structure (is completely buried), and the peptide-binding groove of the PDZ domain exhibits an exquisite complementarity with the shape of the peptide in its bound conformation, surrounding the four C-terminal residues (Figure 6(a)). Indeed, 80% of

the surface of the four C-terminal residues of the ELKS1bC peptide is buried in the complex, whereas 59%, 56% and 67% of the peptide surface areas are buried in the complexes of the third PDZ of PSD-95, the sixth PDZ of GRIP1 and the PDZ domain of nNOS, respectively. The ribbon and stick model shown in Figure 7

Figure 4. Structure of the RIM1a PDZ domain/ELKS1bC peptide complex. (a) Backbone superposition of the 20 structures of the complex with the lowest energies with the RIM1a PDZ domain shown in blue and the ELKS1bC peptide shown in orange. Only residues in the PK4 to P0 positions are shown; the remaining peptide residues are unstructured. N and C indicate the N and C termini, respectively. (b) Ribbon diagram of a representative structure of the RIM1a PDZ domain with the backbone of the ELKS1bC peptide shown as a stick model. The elements of secondary structure of the RIM1a PDZ domain are labeled. The models were prepared with Pymol (DeLano Scientific, San Carlos, CA; http://pymol.sourceforge.net/).

460

Solution Structure of the RIM1a PDZ Domain

Table 1. Structure statistics for the 20 structures of the RIM1 PDZ domain/ELKS1bC peptide complex with the lowest energies Average r.m.s. deviations from experimental restraints (1568 total) ˚) NOE distance restraints (A All Intraresidual (iZj) Sequential (jiKjjZ1) Short range (jiKjjZ2–4) Long range (jiKjjO4) Intermolecular ˚) Hydrogen bond (A Torsion angle restraints (deg.) RDCs (Hz) Average r.m.s. deviations from idealized covalent geometry ˚) Bond lengths (A Bond angles (deg.) Improper angles (deg.) Ramachandran plot statisticsa Most favored region (%) Additionally allowed region (%) Generously allowed region (%) Non-allowed region (%) ˚) Average r.m.s. deviations from atomic coordinates (A Overall backbone (7–114,186–191) Overall heavy atom (7–114,186–191) Ordered backbone (secondary structure)b Ordered heavy atom (secondary structure)

1280 156 422 200 502 73 66 149 73

0.0095G0.0002 0.0064G0.0013 0.0094G0.0004 0.0127G0.0005 0.0088G0.0004 0.0062G0.0021 0.228G0.016 1.00G0.06 0.0017G0.00005 0.381G0.007 0.21G0.01 83.7 14.2 1.6 0.5

Among 20 structures 0.95 1.64 0.36 0.93

To average structure 0.70 1.13 0.27 0.64

˚ or dihedral angle violation larger All 20 structures have NOE energies below 10 kcal/mol. There was no NOE violation larger than 0.2 A than 58. a Calculated with PROCHECK.54 b The structures were superimposed using the backbone atoms of residues in secondary structure elements (9–12, 19–25, 41–45, 56– 61,66–69,79–82, 85–86, 92–103, 108–112, 188–191).

gives a more complete view of the side-chains from the RIM1a PDZ domain that contact the ELKS1bC peptide. These side-chains are indicated with a star in the sequence alignment shown in Figure 8, which includes the PDZ domains of several vertebrate and invertebrate RIMs, the rat piccolo PDZ domain, and some prototypical PDZ domains with different specificities. It is apparent in this sequence alignment that most of the side-chains from the RIM1a PDZ domain involved in peptide recognition are

highly conserved in RIMs. Some of these sidechains are conserved selectively in RIMs (highlighted in blue in Figure 8), while others are conserved in PDZ domains in general (highlighted in red). It is noteworthy also that almost all sidechains involved in ELKS-binding are identical in the RIM1a and RIM2 PDZ domains, which is not surprising, given the fact that they exhibit the same binding specificity.20 The binding pocket for the alanine residue in the

Figure 5. Superpositions of ribbon diagrams of the RIM1a PDZ domain (blue) with the piccolo PDZ domain (a) and the third PDZ domain of PSD-95 (b) (orange). The PDB accession numbers of the piccolo PDZ domain and the third PDZ domain of PSD-95 are 1ujd and 1be9, respectively. The models were prepared with Pymol (DeLano Scientific, San Carlos, CA).

Solution Structure of the RIM1a PDZ Domain

461

Figure 6. The ELKS1bC peptide binds to a narrow and deep groove of the RIM1a PDZ domain. (a)–(d) Surface representations of (a) the RIM1a PDZ domain, (b) the third PDZ domain of PSD-95, (c) the sixth PDZ of GRIP1 and (d) the PDZ domain of nNOS, complexed with target peptides (the PDB accession numbers are 1be9, 1n7f and 1b8q for (b)–(d), respectively). The surfaces of the PDZ domains are represented in blue, and the bound peptides are shown as stick models with the P0, PK1, P-K2, and PK3 residues colored pink, red, yellow and orange, respectively. The side-chains that form the top surface of the groove in the RIM1a PDZ domain are labeled in (a). The models were prepared with Pymol (DeLano Scientific, San Carlos, CA; http://pymol.sourceforge.net/).

P0 position of the ELKS1bC peptide is formed primarily by the side-chains of L629, L630, L632 and W673 from the RIM1a PDZ domain (Figure 7). Among these, the bulky W673 side-chain is

Figure 7. The RIM1a PDZ domain/ELKS1bC peptidebinding mode. The Figure shows a ribbon diagram of the RIM1a PDZ domain with the side-chains that contact the ELKS1bC peptide displayed as blue stick models and the ELKS1bC peptide shown as a stick model with the same color-coding as Figure 6. The model was prepared with Pymol (DeLano Scientific, San Carlos, CA; http:// pymol.sourceforge.net/).

conserved selectively in RIMs (Figure 8) and is likely critical for the strict requirement for the small alanine side-chain in the ELKS P0 position. On the other hand, L630 is highly conserved in PDZ domains, while L632 is highly conserved in RIM PDZ domains and is usually replaced by other hydrophobic residues in other PDZ domains. L629 provides a lid that helps to bury the alanine residue of the ELKS1bC peptide. Although this residue is variable in RIM PDZ domains, the different sidechains in this position may still be able to cover the C-terminal ELKS residue in a fashion similar to that observed for the RIM1a PDZ domain/ELKS1bC peptide complex. The tryptophan side-chain in the PK1 position of the ELKS1bC peptide is substantially more buried than those of most PDZ domain targets (Figure 6), and is surrounded by L629 and three lysine sidechains of the RIM1a PDZ domain (K633, K651 and K653). These three lysine side-chains are conserved selectively in RIM PDZ domains (Figure 8) and are thus likely to be critical for recognition of the tryptophan side-chain in the PK1 position, particularly the K633 and K651 side-chains. The mode of recognition of the ELKS tryptophan is somewhat similar to that observed in the Erbin PDZ domain, which was shown to have a high selectivity for a tryptophan in the position PK1 of target peptides using a combinatorial phage display approach.39 The hydrophobic isoleucine side-chain in the PK2 position of the ELKS1bC peptide is characteristic of class II PDZ domain-binding motifs and, correspondingly, binds to a hydrophobic pocket formed

462

Solution Structure of the RIM1a PDZ Domain

Figure 8. Sequence alignment of RIM PDZ domains from different species, the piccolo PDZ domain, and prototypical examples of class I, II and III PDZ domains. Residues conserved in most PDZ domains displayed in the alignment (O80% of the sequences; EZD, KZR, NZQ, LZVZIZM, FZY, and SZT) are shown in white with a red background. Residues that appear to be conserved selectively in RIM PDZ domains are shown in white with a blue background. Residues that are conserved in other PDZ domains, but are distinct in RIM PDZ domains, are shown in black with a yellow background. The secondary structure elements of the RIM1a PDZ domain are illustrated above the alignment, and residues from the RIM1a PDZ domain that contact the ELKS1bC peptide are indicated by asterisks (*). The third PDZ domain of PSD-95 and the PAR-6 PDZ domain belong to class I, the INAD PDZ domain and the sixth PDZ domain of GRIP1 belong to class II, and the nNOS PDZ domains belongs to class III. Species abbreviations: RN, rat (Rattus norvegicus); HS, human (Homo sapiens); Am, honey bee (Apis mellifera); DM, fruit-fly (Drosophila melanogaster); CE, worm (C. elegans). The GI numbers for the sequences displayed are: RIM1 Rn, 34395745; RIM2 Rn, 34395746; Unc10 Ce, 41017531; RIM Am, 66517720; RIM Dm: 61679350; Piccolo Hs, 42543726, PSD95 third Rn, 3891677; PAR-6 Dm, 42543681; INAD Dm, 15826741; GRIP1 sixth Rn: 34810638; nNOS Rn, 4929899.

by the V686, Y687, Y690, L691 and K694 of the RIM1a PDZ domain. However, this binding pocket is deeper than those commonly observed in class II PDZ domains (e.g. compare Figure 6(a) with (c)), in large part because of the bulky Y687 side-chain. This is the only one among the four hydrophobic residues involved in contacts with the isoleucine side-chain in the PK2 position that is conserved selectively in RIM PDZ domains (Figure 8), and is thus likely to be critical for recognition of this side-chain. It is important to note also that the RIM1a PDZ domain does not conform to the common pattern of PDZ domains, whereby the first residue of helix aB facing the peptide-binding groove is critical to determine the specificity for the PK2 position (e.g. histidine in class I PDZ domains and a hydrophobic residue in class II PDZ domains).40 In the RIM1a PDZ domain, this role is played by the Y687 side-chain, while the first residue of helix aB pointing toward the peptide binding groove (N683) does not contact the peptide. This distinction is made possible by the fact that helix aB of the RIM1a PDZ domain is longer and partially displaced with respect to the usual position of helix aB in PDZ domains (Figure 5(b)). Note also that the

Y687 side-chain is not conserved in the piccolo PDZ domain, which likely underlies, in part, its distinct specificity from the PDZ domain of RIMs (see Discussion). The Y687 side-chain also dictates, together with the K633 side-chain, the narrow nature of the region of the groove of the RIM1a PDZ domain where the glycine residue at the PK3 position of the peptide binds (see Figure 6(a)). This residue also contacts the backbone and side-chain of V635 from the RIM1a PDZ domain, which forms the bottom of the groove in this region. Even an alanine side-chain in the PK3 position would cause steric clashes with the backbone atoms at the bottom of the groove. This feature, together with the shape complementarity between the glycine atoms and the groove formed by K633, V635 and Y687, most likely accounts for the requirement for glycine at the PK3 position (even a Gly to Ala mutation abolishes binding).20

Discussion PDZ domains are widespread protein modules

Solution Structure of the RIM1a PDZ Domain

that function in protein–protein recognition and help to organize large macromolecular assemblies involved in diverse signaling processes. Many PDZ domains exhibit similar specificities that have been categorized into different classes according to the sequence characteristics of their target peptides, but an increasing number of PDZ domains have been found to exhibit unusual specificities that do not conform to any of these common classes.1–4 Characterizing the modes of interaction between these PDZ domains and their targets is important for understanding the structural basis for their unusual specificity, and for gaining a better view of the determinants of PDZ domain specificity in general. The RIM PDZ domain constitutes an intriguing example of a PDZ domain that exhibits a unique specificity for the ELKS C-terminal sequence20 in a protein that plays key roles in regulating neurotransmitter release and organizing the presynaptic active zone.22–26 Our results show that the affinity of the RIM1a/ELKS interaction is significantly higher than those commonly observed for other PDZ domain/target interactions and further emphasize this specificity, showing that the closely related PDZ domain of piccolo does not bind to the ELKS C-terminal sequence. Moreover, the 3D structure of the RIM1a PDZ domain/ELKS1bC peptide complex described here shows how the specificity of target recognition by the RIM1a PDZ domain is achieved by formation of an unusually narrow and deep groove with an exquisite shape complementarity to the four ELKS C-terminal residues in their bound conformation. The importance of the side-chains in the P0 and PK2 positions of target peptides for specific binding to PDZ domains was established early,7,8 and could easily be rationalized by the finding that the peptides bind in an extended conformation that places these two side-chains on the same side of the peptide, facing the PDZ domain surface.9,11 However, multiple examples showed that other C-terminal residues could also determine binding specificity.1–4 For instance, the second PDZ domain of MAGI3 and the Erbin PDZ domains offer examples where a tryptophan residue in the PK1 position of the target is critical for high-affinity binding,39,41 and a threonine residue in the PK3 position contributes to the specificity of target binding to the GRIP1 PDZ domain.38 What is unusual in the RIM PDZ domain/ELKS interaction is the critical importance of each of the four ELKS C-terminal residues for binding.20 The structure of the RIM1a PDZ domain/ ELKS1bC peptide complex shows that binding of these four C-terminal residues occurs at a deep and narrow groove that contrasts with the shallower surfaces commonly involved in binding of PDZ domains to their targets (see Figure 6). Such shallow surfaces allow substitution of the more exposed side-chains with little effect on binding, while the narrow groove of the RIM1a PDZ domain includes four deep,

463 well-defined binding pockets that dictate the specific requirement for each of the four residues in the ELKS C-terminal sequence. Formation of this groove undoubtedly depends on multiple residues of the RIM1a PDZ domain that influence its overall structure, but also on sidechains that contact the peptide and are selectively conserved in RIM PDZ domains (Figure 8). Among these, the K633, K3651, W673 and Y687 side-chains appear to have a preponderant role in determining specificity. Most of the RIM sidechains involved in contacts with the ELKS peptide are conserved in the piccolo PDZ domain, but a conspicuous distinction is the lack of conservation of the RIM Y687 side-chain (which is glutamine in piccolo; see Figure 8). This side-chain is located in helix aB, which is shorter and slightly displaced in the piccolo PDZ domain (see Figure 5(a)). It is most likely that these differences in the length, location and sequence of the aB helix underlie, at least in part, the distinct specificities of the RIM and piccolo PDZ domains. The complementarity between the ELKS C-terminal sequence and the RIM1a binding groove may also favor the relatively strong affinity of their interaction (270 nM dissociation constant according to our ITC measurements). Understanding the biological implications of the high specificity and relatively high affinity of the RIM/ELKS interaction will require mutational and functional analyses in vivo. It seems likely that the specific recognition of ELKS by RIMs may be critical for the proper organization of active zones, which are highly complex assemblies of very large proteins.42 Thus, such specificity is expected to allow interaction of the ELKS C-terminal sequence with the RIM PDZ domain without interference from interactions with the PDZ domain of piccolo, which is likely to be proximally located by virtue of coiled-coil interactions between piccolo and ELKS.36 On the other hand, the relatively high affinity of the RIM/ELKS interaction may be important for ensuring that sufficiently high concentrations of ELKS are accumulated at the active zone, and could help in the proper organization of the active zone. Note that active zones are highly insoluble and are probably held together by a network of protein–protein interactions. Thus, the affinity of the RIM/ELKS interaction may contribute to stabilizing this network. At the same time, binding of ELKS to RIMs is likely to be further strengthened through cooperativity with other protein–protein interactions involving these and other active zone proteins, e.g. the ELKS/piccolo interaction. Verifying the validity of these ideas will require a more thorough characterization of the structural organization of the active zone. The structure of the RIM1a PDZ domain/ELKS1bC peptide complex described here offers a first glimpse at atomic resolution for one of the protein–protein interactions that may contribute to this organization.

464

Materials and Methods Protein expression and purification The construct for expression of the rat Rim1a PDZ domain (residues 596–704) has been described.20 To generate uniformly 15N-labeled or 15 N,13C-labeled samples for NMR studies, bacteria were grown in minimal medium supplemented with 15NH4Cl and with or without [13C6]glucose (CIL, Andover, MA) as the sole nitrogen and carbon sources, respectively. The fusion protein was affinity-purified on glutathione-Sepharose (Pharmacia), cleaved from the glutathione-S-transferase (GST) moiety with thrombin (Sigma) and purified further by ion-exchange and gel-filtration chromatography. Nonlabeled ELKS1bC peptide (CDQDEEEGIWA) was synthesized by the Protein Core Facility of UT Southwestern. To obtain isotopically labeled ELKS1bC peptide, a cDNA fragment encoding its amino acid sequence was inserted between the HindIII and BamHI sites of the pTMHa vector (kindly provided by Peter Kim). The isotopically labeled ELKS1bC peptides were then expressed and purified as described.43 Isothermal titration calorimetry ITC experiments were performed using a VP-ITC system (MicroCal) at 20 8C in a buffer composed of 20 mM Pipes (pH 6.9), 150 mM NaCl and 1 mM Tris(2carboxyethyl)-phosphine (TCEP). The proteins were extensively dialyzed against the buffer, centrifuged and degassed before the experiment. Typically, 200 mM peptide solution was injected 35 times in 8 ml aliquots into the 1.8 ml sample cell containing Rim1a PDZ domain at a concentration of 10–20 mM. Data were fit with a nonlinear least-squares routine using a single-site binding model with Origin for ITC v.5.0 (Microcal), varying the stoichiometry (n), the enthalpy of the reaction (DH) and the association constant (Ka). NMR spectroscopy All NMR experiments were acquired at 30 8C on Varian Inova500 or Inova600 spectrometers using two sets of samples (total of six) of 0.5mM rat RIM1a PDZ domain/ ELKS1bC peptide complex dissolved in 20 mM Mes (pH 6.5), 100 mM NaCl, 1 mM TCEP. The first set of samples (total of three) included 15N-labeled, 15N,13C-labeled or 10% 13C-labeled RIM1a PDZ domain (0.5 mM) mixed with 0.75 mM unlabeled ELKS1bC peptide; the second set of samples (total of three) included 15N-labeled, 15 N,13C-labeled and 10% 13C-labeled ELKS1bC peptide (0.5mM) mixed with 0.75mM unlabeled RIM1a PDZ domain. Resonance assignments and NOE analyses were performed using a suite of pulsed-field gradient enhanced NMR experiments for each set of samples as described.44 Briefly, these included 3D 1H–15N total correlated spectroscopy (TOCSY)-HSQC, HNCO, HNCACB, CBCA(CO)NH, (H)C(CO)NH-TOCSY, H(C)(CO)NH-TOCSY, and HCCH-TOCSY spectra for resonance assignments, and 2D NOESY, 3D 1H–15NNOESY-HSQC, and 3D 1H–13C NOESY-HSQC experiments (100 ms mixing times) to measure NOEs for structure determination.45–48 To obtain unambiguous intermolecular NOEs, we also recorded 3D 1H–13C NOESY-HSQC experiments without decoupling in the t1 dimension, as well as 2D 13C-filtered,13C-edited 2D NOESY-HSQC experiments. Protection of amide protons

Solution Structure of the RIM1a PDZ Domain

from the solvent was measured from the intensities of exchange cross-peaks with the water resonance in the 3D 1 H–15N-TOCSY-HSQC spectra. 1H–15N residual dipolar couplings were measured from a sample partially aligned in 5% (w/v) 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) (3:1; Avanti Polar Lipids) at 35 8C. Stereospecific assignments of Val and Leu methyl groups were obtained from a constant-time 1H–13C HSQC spectra acquired on 10% 13C-labeled samples. All data were processed with NmrPipe,49 and analyzed with NMRview.50 Structure calculations NOE cross-peak intensities were classified as strong, medium, weak, and very weak, and assigned to restraints ˚ , 1.8–3.5 A ˚ , 1.8–5.0 A ˚ and 1.8–6.0 A ˚ , respectof 1.8–2.8 A ively, with appropriate pseudoatom corrections. Phi and psi torsions angle restraints were included based on analysis of HN, 15N, 13Ca, 13CO, and 13Cb chemical shifts using the program TALOS.51 Dihedral angles were restrained to the maximum of 22.58 or 1.5 times the standard deviation observed in the TALOS database matches. Hydrogen bond restraints were set for amide protons protected from exchange with 2H2O solvent with ˚ !dNH–O!2.5 A ˚ and 2.5 A ˚ !dN–O!3.5 A ˚ restraints. 1.5 A The initial structures of the complex were calculated without the residual dipolar coupling restraints and refined using torsion angle simulated annealing with CNS.52 Determination of alignment tensor parameters (Da and R) was carried out using PALES,53 and the initial models calculated above. The residual dipolar coupling restraints were then incorporated into the final CNS calculations. Structural statistics were calculated with CNS and PROCHECK.54 Protein Data Bank accession code The structures of the RIM1a PDZ domain/ELKS1bC peptide complex have been deposited in the Protein Data Bank with accession code 1ZUB.

Acknowledgements J.L. was a postdoctoral fellow from the American Heart Association. This work was supported by NIH grants NS40944 and NS37200 to J.R.

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Solution Structure of the RIM1a PDZ Domain

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Edited by M. F. Summers (Received 30 May 2005; received in revised form 13 July 2005; accepted 14 July 2005) Available online 10 August 2005