Ligand-dependent Dynamics and Intramolecular Signaling in a PDZ Domain

doi:10.1016/j.jmb.2003.11.010

J. Mol. Biol. (2004) 335, 1105–1115

Ligand-dependent Dynamics and Intramolecular Signaling in a PDZ Domain Ernesto J. Fuentes1,4, Channing J. Der2,4 and Andrew L. Lee1,3,4* 1 Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill Chapel Hill, NC 27599, USA 2

Department of Pharmacology University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA 3 Division of Medicinal Chemistry and Natural Products, School of Pharmacy University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA 4 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill Chapel Hill, NC 27599, USA

Allosteric communication is a fundamental process that proteins use to propagate signals from one site to functionally important distal sites. Although allostery is usually associated with multimeric proteins and enzymes, “long-range” communication may be a fundamental property of proteins. In some cases, communication occurs with minimal structural change. PDZ (post-synaptic density-95/discs large/zonula occludens-1) domains are small, protein –protein binding modules that can use multiple surfaces for docking diverse molecules. Furthermore, these domains have long-range energetic couplings that link the ligand-binding site to distal regions of the structure. Here, we show that allosteric behavior in a representative member of the PDZ domain family may be directly detected using side-chain methyl dynamics measurements. The changes in side-chain dynamics parameters in the second PDZ domain from the human tyrosine phosphatase 1E (hPTP1E) were determined upon binding a peptide target. Long-range dynamic effects were detected that correspond to previously observed pair-wise energetic couplings. These results provide one of the first experimental examples for the potential role of ps – ns timescale dynamics in propagating long-range signals within a protein, and reinforce the idea that dynamic fluctuations in proteins contribute to allosteric signal transduction. q 2003 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: protein dynamics; allostery; PDZ domain; NMR; protein– protein interaction

Introduction Proteins “sense” effector ligands through modulated internal energetics that are manifested in structural and dynamic properties. Often, such molecular signals are transmitted over long distances, linking disparate sites within or between protein subunits. Although allostery is usually associated with multimeric proteins and enzymes, “long-range” communication may be a fundamental property of proteins. In some cases, communication occurs with minimal structural change. Examples include staphylococcal nuclease,1 myoglobin,2 serine proteases,3 HIV-1 Abbreviations used: PDZ, post-synaptic density-95/ discs large/zonula occludens-1; hPTP1E, human protein tyrosine phosphatase 1E; NMR, nuclear magnetic resonance; RA-GEF2, Ras-associated guanine nucleotide exchange factor 2; PLC-g1, phospholipase C-g1; CaM, calmodulin. E-mail address of the corresponding author: [email protected]

protease,4 dihydrofolate reductase,5 – 7 b-lactamase,8 and PDZ3 of PSD-95.9 Equilibrium thermodynamic evidence for the interdependence of two or more sites can be obtained through double-mutant cycles, by showing that the energy of interaction ðDGint Þ is nonzero.10,11 A non-zero DGint value indicates that the sites are thermodynamically coupled. Knowledge of such pair-wise interactions can lead to an appreciation for the relative contribution of each residue towards protein function and/or stability. An alternative approach to identifying residue – residue couplings has been suggested, whereby functionally important residues are identified based on statistical analysis of a large and diverse multiple sequence alignment of a conserved protein family.9,12 When applied to the PDZ (postsynaptic density-95/discs large/zonula occuldens1) domain family, “statistical couplings” predict thermodynamically coupled residue pairs.9 Most interesting is the finding that energetically coupled residues are physically linked, forming a “conserved energetic pathway” from a functionally

0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

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important residue to distal residues in the protein. The precise structural and dynamic nature of this information transmission has not yet been clearly delineated, thus providing motivation for the work described here. The PDZ domain has been considered a passive, protein –protein binding domain, but it is becoming increasingly evident that it is significantly more diverse.13 – 15 PDZ domains are protein –protein interaction domains , 100 amino acid residues long that bind the C-terminal four to six residues of their target protein. More recently it has been shown that amino acid sequences corresponding to internal PDZ-binding motifs can also serve as ligands.16 Novel modes of ligand binding have been described for GRIP1 PDZ717 and Par618 PDZ domains. For Par6, this binding is regulated allosterically.19 Hence, PDZ domains have the capacity to use multiple surfaces for docking a diversity of molecules, and these binding events can be allosterically regulated. Here, we show that nuclear magnetic resonance (NMR) spin relaxation measurements may be used to directly detect intraprotein, allosteric signals. We performed deuterium-based methyl sidechain dynamics analyses on a PDZ domain derived from the human protein tyrosine phosphatase 1E (hPTP1E). These studies were carried out on the free PDZ protein, as well as bound to a C-terminal peptide derived from a cognate-binding protein. hPTP1E is a soluble protein tyrosine phosphatase containing multiple PDZ domains that mediates a range of diverse biological processes such as apoptosis,20,21 cytokinesis,22 and signaling.23 The solution structure of the second PDZ domain (PDZ2) in the absence of ligand has been solved for the mouse24 and human homologue.25 In addition, human PDZ2 in complex with a C-terminal peptide ligand derived from the Ras-associated guanine nucleotide exchange factor 2 (RA-GEF2) has been solved.26 Consistent with all PDZ domain structures, the topology of PDZ2 consists of two a-helices and six b-strands arranged in a b-barrel fold (Figure 1A). PDZ2 binds the RA-GEF2 peptide in a manner similar to other PDZ domain/peptide complexes.13 An extended groove between strand b2 and helix a2 of the PDZ domain forms a pocket,allowing the RA-GEF2 peptide to insert by b-strand addition.27 The peptide participates in various backbone hydrogen bonds with strand b2, typical of the bonding pattern normally seen in b-sheets. Additional hydrogen bonds are made to the peptide ligand by residues in helix a2 and residues within the b1/b2 loop of PDZ2.26 This “carboxylate-binding” loop is critical for binding the free carboxylate of the C-terminal hydrophobic residue of the peptide ligand.27 Of further interest in the PDZ2 –RA-GEF2 complex is the side-chain interaction between His71 in helix a2 and the hydroxyl of Ser(2 2) (the peptide ligand is numbered 0 to 2 8 beginning at the C terminus) of the peptide, which defines this PDZ domain as a class I type.14 The RA-GEF2 peptide binds PDZ2 with a

Dynamics of hPTP1E PDZ2

Kd of , 10 mM (data not shown) and is in fast exchange on the NMR timescale.

Results and Discussion Dynamic changes at the peptide-binding site We have used side-chain28 (2H-methyl) and backbone29 (15N) NMR spin relaxation methods to probe the internal dynamics of human PDZ2, free and bound to the RA-GEF2 peptide (Ac-ENEQVSAV-COOH). The degree of angular restriction on the ps – ns timescale for individual bonds is described by the “model-free” generalized order parameter ðS2 Þ that ranges from 0 to 1, corresponding to isotropic disorder and a fixed orientation in the molecular frame, respectively.30,31 For methyl groups, the order parameter ðS2axis Þ used corresponds to the 3-fold methyl symmetry axis that aligns with the C – CH3 bond. The associated timescale of this motion can be obtained from the effective internal correlation time ðte Þ; which is dominated by methyl rotation about the symmetry axis. The backbone and side-chain dynamics for the free and peptide-bound PDZ2 are typical of a well-folded, native protein. The 15N-derived NH S2 values, which report on backbone motions, average , 0.9 in both the bound (Figure 2) and free states24 (data not shown). In contrast, the range of S2axis extends from , 0.2 to , 0.8 (Figure 3A) and averages , 0.6 for all methyl groups in both the free and peptide complexed states. This distinct behavior between side-chain and backbone dynamics has been previously noted.32,33 Figure 3B and C show S2axis values of the free and peptidebound PDZ2 mapped onto the PDZ2 structure. As has been reported by others,34 the S2axis values of methyl moieties are heterogeneously distributed throughout the structure. In this regard, the PDZ2 methyl dynamics in the free or peptide-bound state appear ordinary. Analysis of the data in Figure 3 also highlights the difficulties with interpretation of side-chain dynamics in terms of physical parameters, as no correlation with accessible surface area or packing is seen. The changes in side-chain dynamics upon binding the RA-GEF2 peptide are shown in Figure 4. Of the 41 methyl groups that were compared, nine and 14 showed significant changes in S2axis (Figure 4A) and te (Figure 4B), respectively, indicating that only a small number of side-chain positions have a dynamic response to peptide-binding. This set represents 14 of the 37 total residues containing methyl groups. Of these 14 residues, six are directly involved in peptide-binding and eight are ˚ away from the site of ligand-binding (i.e. . 7 A from the nearest peptide atom). At the peptide-binding site, many affected sidechains have increased rigidity. The pocket that accommodates the C-terminal Val(0) side chain of the RA-GEF2 peptide (Figures 4 and 5A) is formed

Dynamics of hPTP1E PDZ2

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Figure 1. Mouse and human solution structures of the second PDZ domain of PTP1E. A, The average structure derived for the human PDZ2 bound to the RA-GEF2 peptide26 with the elements of secondary structure labeled. B, Representative structural models for the free and peptide-bound PDZ2 protein. The mouse isoform is colored red (PDB code 1GM1)24 while the human form (PDB code 1D5G)26 is colored blue. The RA-GEF2 peptide bound to the human PDZ2 is shown in green. The superposition is based on the published elements of secondary structure. Superposition and calculation of the average structure was performed using MOLMOL.60

by residues Leu18, Ile20, Val22, and Leu78. Leu78d1 has the largest change in S2axis ; , 0.3. In the absence of peptide, its S2axis is , 0.45, which is close to the average for this methyl type.35,36 Upon binding the peptide, both the d1 and d2 methyl groups approach an S2axis of 0.8. In addition, Leu18 and Ile20, both located in the carboxylate-binding loop, and Val22 show changes in te upon peptide binding. Leu18 and Ile20 make hydrophobic interactions with the methyl groups of the peptide C-terminal Val(0) and their NHs contribute to ligating its free carboxylate through hydrogen bonding interactions.26 In the case of Ile20, a small increase in S2axis occurs upon peptide-binding. Finally, both methyl groups of Val22 have changes in te ; and its NH engages in hydrogen bonding with the backbone carbonyl of Ser(2 2) in the peptide.26 Although the precise physical interpretation of te is not straightforward when using the 2H-methyl approach,37 it is a robust

indicator of dynamic perturbations. Because te is determined with high precision, relatively small changes can be detected. Slow (ms – ms) conformational dynamics were assessed from 15N relaxation data for the free and peptide-bound states of PDZ2. Statistically elevated transverse relaxation rates ðR1r Þ were identified for several amide nitrogen atoms along the backbone,38 and model-free fitting39 indicates that an additional relaxation contribution from conformational exchange ðRex Þ40 is needed to fit these data. Of interest are residues in the b1/b2 loop (residues 13 –19) and the b2/b3 loop (residues 24– 35), both regions important for peptide binding.26 In the absence of peptide, residues Asp15 and Asn16, located in the b1/b2 loop, have moderate Rex terms (Figure 2). Residues Ser17 and Leu18 have very weak NH cross-peaks, suggesting conformational exchange, but these data could not be analyzed quantitatively. Residues Asn27, Thr28, Arg31, and Gly34 in b2/b3 loop have

Figure 2. Slow (ms– ms) time scale motions are damped upon peptide-binding. 15N-backbone order parameters, S2 (filled circles), for the PDZ2– RA-GEF2 peptide complex, and fitted Rex terms (see Materials and Methods) for the free (filled triangles) and peptide-bound (open triangles) PDZ2 are plotted as a function of primary sequence.

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Dynamics of hPTP1E PDZ2

Figure 3. Side-chain order parameters for free and peptide bound hPTP1E PDZ2. A, A plot of the derived S2axis parameters for free (black) and peptide-bound (gray) PDZ2 as a function of methyl group. B and C, The S2axis parameter mapped onto the hPTP1E PDZ2 structure.26 Methyl groups are color-coded from white to dark blue on a continuous scale, corresponding to S2axis ranging from 0 ! 1. Methyl groups in yellow were not analyzed.

Figure 4. Dynamic response of PDZ2 to peptide binding. A, The difference in S2axis parameter, DS2axis (bound-free) upon peptide binding and B, the difference in internal correlation time, Dte (bound-free) upon peptide binding. Methyl groups with significant changes are shaded black. Asterisks denote residues that interact with the peptide ligand.

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Figure 5. Comparison between dynamically linked residues in PDZ2 and thermodynamic couplings predicted from the family of PDZ domains. A, A summary of residues whose dynamical parameters changed significantly upon peptide-binding mapped onto the structure of PDZ2– RA-GEF2.26 The PDZ2 protein secondary structure is colored gray while the peptide ligand is green. Red VDW surfaces are side-chain methyl residues that had appreciable DS2axis ; in yellow are those methyl residues with appreciable Dte ; and in blue are backbone NHs that displayed DRex : Methyl groups not having changes in dynamics parameters are represented as gray VDW surfaces. Residues that were not compared are shown in black. For clarity, three views are presented, incremented by 908 rotations. Residues of importance are labeled. B, The statistical couplings derived from Lockless & Ranganathan9 mapped onto PDZ2 – RA-GEF2, shown in identical views as in A. C, The primary sequence alignment of four PDZ domains: hPTP1E PDZ2, PSD-95 PDZ3, rGRIP1 PDZ7, and mPar6 (Swiss-Prot accession no.: Q12923, P31016, P97879, and Q9JK83). Italicized residues in hPTP1E PDZ2 summarize the dynamics results presented here, and are color-coded as in A. Italicized residues in the PSD-95 sequence summarize the results of Lockless & Ranganathan.9 Italicized residues in the remainder of sequences are important for ligand-binding. The boxed sequences are generally important for ligand-binding in class I PDZ domains.

significant Rex terms that are also present in the free state of the mouse homologue.24 In all cases, however, Rex terms are damped upon peptidebinding (Figure 2). Consistent with this Rex damping, the side chains of Val26 and Val30 have increased methyl S2axis values in the presence of peptide and parallel changes in te (Figure 4). The change in S2axis in Val26 can be rationalized by the new set of interactions formed between residue Glu(2 5) of the RA-GEF2 peptide with the backbone NH of Asn27 and the side-chain of Val26.26 The case for Val30 is less clear because this residue does not directly participate in peptide-binding. Taken together, the side-chain dynamics and backbone-detected Rex data suggest an overall, classical rigidification of the ligand-binding site upon peptide-binding. This is highlighted in the b2/b3 loop region, in which loss of motion on the ps –ns and

ms – ms timescales occurs coordinately. The spatial proximity of the residues containing these different timescale motions and their concurrent disappearance upon peptide binding may point towards a hierarchy of dynamical processes in ligand-free PDZ2. Finally, we emphasize that the most significant ps – ns motions and their changes are observed at the side-chain level. These observations are similar to those reported for other protein– peptide complexes.32,33,41,42 In particular, the C-terminal SH2 domain of phospholipase C-g1 (PLC-g1) complexed to a phosphopeptide32 showed changes in side-chain dynamics of similar magnitude ðDS2axis # 0:1 – 0:15Þ but for a larger number of residues, most of which were at the protein –peptide interface. Moreover, this complex also showed a correspondence between changes in Rex and S2axis values in residues at the

1110 binding interface.29,32 The complex between calmodulin (CaM) and the smooth muscle myosin light-chain kinase peptide (smMLKCp) showed larger changes in dynamics ðDS2axis . 0:2Þ: The CaM – peptide complex has a surface area of ˚ 2, while the PDZ2 complex has a smaller , 2000 A ˚ 2, which is comparable binding interface of , 430 A in magnitude to that seen in the PLC-g1 SH2 com˚ 2). These differences in dynamic plex (, 600 A response and interaction area may represent different binding strategies,33 and may also scale with overall binding affinities (or inversely with koff ). Qualitatively, they correlate with the protein – peptide affinity for each complex: CaM , 1 nM33 . PLC-g1 , 0.10 mM32 . PDZ2 , 10 mM (unpublished results), suggesting that the dynamic response observed for side chains, especially at the binding interface, may be linked to binding energetics. Thus, an increased binding affinity may provide the driving force necessary to produce larger dynamic responses to ligand binding. The peptide-binding site is dynamically linked to distal sites Of central interest are the eight residues that display significant changes in ps – ns side-chain dynamics parameters, but map to two distinct regions in PDZ2, each distal from the peptidebinding site. The first region (“distal surface 1”) encompasses residues in the N terminus of b6 and the anti-parallel b-sheet element formed by b4 and b5 (Figures 5 and 6). Both methyl groups of ˚ from the C-terminal peptide Val85, located , 9 A residue (Val(0)), show a decrease in S2axis upon pep-

Figure 6. Distal surfaces in PDZ2 are physically linked to the ligand-binding site. A diagrammatic representation of the PDZ1 –RA-GEF2 structure (in the 08 orientation of Figure 5A) is shown with three shaded surfaces, each defined by residues having perturbed dynamics upon peptide binding. Two distal surfaces that are in close proximity to the peptide-binding site are depicted. Distal surface 1 is comprised of Thr81, Val85, Val61, Val64, Leu66, and Ala69. Distal surface 2 contains Ala39 and Val40. The peptide-binding site contains residues that interact with the peptide and have perturbed dynamics (see the text).

Dynamics of hPTP1E PDZ2

tide binding. Likewise, Val61g1, Val64g2, and Ala69b show changes in side-chain S2axis and te values. ˚ away from the closest These residues are , 11 A residue of the RA-GEF2 peptide. Thr81g2 and Leu66d1 do not exhibit changes in S2axis but have significant changes in te : Figures 5A and 6 show that despite not being directly involved in peptide binding, Val85, Thr81, Val61, Val64, Leu66, and Ala69 are physically linked by van der Waals interactions to form distal surface 1. Distal surface 1 is linked to the peptide-binding site through residue Leu78, which bridges the peptide binding to Thr81 and Val61. A second region distal from the binding site (“distal surface 2”) also shows changes in dynamics. This region is located adjacent to helix a1 and is comprised of residues Ala39 and ˚ from the RAVal40. These residues are , 11 A GEF2 peptide. They show significant changes in te and form a continuous van der Waals surface with Ile20, which itself is directly involved in peptide binding. One might envision that upon peptide binding, residues directly involved in the binding interaction (including the peptide itself) efficiently transduce binding energy through the protein matrix to distal regions via a network of linked interactions (Figure 6). This energy is not randomly dissipated, but instead is directed to specific regions of the protein that are linked to peptide binding. This is clearly seen in Figures 4 and 5A, in which only a subset of residues respond to peptide-binding, and the face opposite the peptidebinding site shows no appreciable changes in dynamics. Protein dynamics correlate with thermodynamic couplings in the PDZ family In a study by Lockless & Ranganathan,9 residue –residue “statistical couplings” were identified in the PDZ domain family from a multiple sequence alignment. When mapped onto the structure of a representative PDZ domain (Figure 5B), the full set of couplings physically link His71 (hPTP1E PDZ2 numbering) at the peptide-binding site to residues throughout the protein, including various residues essential for peptide binding. Interestingly, a number of residues distal from the peptide-binding site were also coupled to His71, resulting in an “energetic communication pathway”. This result and more recent work12,43 suggest that proteins contain evolutionarily conserved “sparse networks” that physically link functionally distant sites in proteins. It is important to note that this result comes from the analysis of a large number of PDZ domain sequences, and not from an individual PDZ domain. Thus, communication pathways observed for the family only represent those shared by the majority of PDZ domains, and individual PDZ domains may contain varied or additional pathways. Nevertheless, as was shown for PDZ3 of PSD-95, the majority of the predicted statistical couplings were in fact thermodynamically coupled.9 It should also be noted that this

Dynamics of hPTP1E PDZ2

previous study identified sites coupled only to His71. Here, we have perturbed the entire hPTP1E PDZ2-binding site (including His71) by binding of RA-GEF2 peptide, and monitored the dynamic response. For these reasons (and others, see below), a perfect correlation between the statistical couplings and our dynamic response is not expected. However, if ps –ns dynamics play a role in or are affected by long-range communication, one would expect to see similarities in the response patterns. Despite the different approaches taken, the pattern of residues that undergo changes in dynamics upon peptide-binding in PDZ2 bears a striking resemblance to the pattern of statistical couplings.9 These dynamic and statistical coupling “responses” are mapped onto the three-dimensional structure of PDZ2 –RA-GEF2 (Figure 5A and B) and highlighted in the amino acid sequence of hPTP1E PDZ2 (dynamics) and PSD-95 PDZ3 (statistical couplings) (Figure 5C). Residues at the peptide-binding site and distal surface 1 comprise the majority of this correlation. Of the 12 residues found to be statistically coupled only six could be directly compared to our dynamics data because only methyl-containing amino acid residues can be compared. Residues Ile20, Val85 and Val61 (V75 is overlapped in our spectra) all correlate with the statistical couplings. Furthermore, residues on the opposite face of the binding site (1808 view) have no changes in dynamics parameters upon peptide binding and show no statistical couplings. The most interesting feature is the correlation between side-chain dynamics and energetic couplings for Val85 and Val61, which form the core of distal surface 1. These residues, although spatially distant from the peptide-binding site ˚ ), sense peptide binding on a dynamic and, (, 11 A therefore, energetic level. Based on the statistical couplings,9 Ala46 and Ile52 (located in helix a1) might be predicted to be dynamically coupled to peptide binding. We do not see dynamic changes in these residues, although changes are observed in Ala39 and Val40, which comprise distal surface 2 and neighbor Ala46 and Ile52. The connectivity to helix a1 may not be optimized in PDZ2; other PDZ domains may show a more complete dynamic and energetic connection to residues in helix a1. In summary, the correspondence between dynamic changes and statistical couplings indicate that side-chain dynamics studies may serve to identify distal regions important for protein function. These observations are also consistent with a role of fast (ps – ns) protein dynamics in the propagation of long-range allosteric signals in PDZ2, and may represent a more general phenomenon in PDZ domains. Our data show that upon peptide binding there is a significant change in PDZ2 dynamics. With respect to structural changes, other PDZ domains show changes in structure along the backbone ˚ upon ligand-binding on the order of 0.7– 1.1 A RMSD.27,44 Direct comparison between the human

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PDZ2 free and peptide-complexed state is complicated by uncertainties in the free structure as pointed out by Walma et al.24 Consequently, the solution structure of the free mouse homologue24 was used for structural comparisons (the mouse and human PDZ2 protein are , 94% identical). From the comparison of the free mouse PDZ2 and the human PDZ2 complexed to the RA-GEF2 pep˚ tide, the overall difference in structure is , 1.2 A RMSD along the six b-strands. The largest structural changes occur at the protein –ligand interface (Figure 1B) at strand b2 and helix a2. However, at the locations where long-range changes in dynamics occur (Val85 and Val61), there are no significant structural changes. Although it is likely that changes in structure contribute to the observed dynamic perturbations at the binding site, the redistribution of dynamics distally appears to result from a cascade of motional fluctuations that occur within a conformational state. Analyses of other protein –peptide complexes also provide evidence for long-range dynamic effects. In the PLC-g1 – peptide complex both methyl groups of Leu35 had large changes in S2axis ˚ from the peptide ligand.32 (, 0.15), yet are , 13 A Data from the CaM – smMLCKp complex indicates that residues near each EF hand pair have per˚ from turbed side-chain dynamics, yet are , 13 A the smMLCK peptide.33 In the 2H-methyl dynamics study of the Ras family guanosine-50 -triphosphotase (GTPase) Cdc42 bound to the effector domain from p21-activated kinase (Cdc42/p21), appreciable changes in S2axis occurred away from the peptide-binding site, in many cases over distances ˚ .42 Long-range dynamic much greater than 10 A effects are not specific to protein –peptide complexes, as a recent study comparing side-chain dynamics of the wild-type protein L with two single-site variants also indicates dynamic effects ˚ from the site of mutation.45 . 10 A Our results are consistent with the idea that changes in tightly coupled dynamic modes can be used to transduce energetic signals to distal regions on a protein, i.e. allostery. This proposal was formally introduced by Cooper & Dryden,46 whereby a plausible model was formulated for allostery based on the redistribution of dynamic fluctuations about the mean position in a protein structure. In this model, very small changes in the mean positions of many atoms could result in energies of , kT; which is sufficient for allosteric activation. The data presented here represent a vivid example of this behavior, predicted nearly 20 years ago. This model now appears even more plausible, as the amount of residual entropy in proteins is known to be quite large.33,42,47 – 50 As discussed by Wand,34 the large residual entropy raises the possibility that proteins might use changes in dynamic modes to achieve allostery. Thus, the parsing of energy from one site on a protein to another could in principle be coupled to dynamical processes. This dynamic contribution does not rule out the common paradigm associating conformational

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changes with allostery; it merely enriches the possibilities that nature has at its disposal. Implications for biological function/ allosteric mechanism Despite the fact that PDZ domains are most often considered passive protein– protein binding domains, they have more varied and complex functions.14,15 NMR studies of PDZ7 from GRIP1 indicate that a novel hydrophobic surface, comprising residues in the b4/b5 loop and helix a2, can be used for protein –protein interactions.17 This region is coincident with one of the surfaces found here (distal surface 1) to be dynamically coupled to the “classic” peptide-binding site (see Figure 5C for specific interactions). No classic C-terminal peptide ligand has been identified for this PDZ domain, and based on structural considerations it remains unclear if it has the potential to bind a C-terminal peptide.17 Par6 is an adapter protein that contains a semiCRIB (Cdc42/Rac1 interactive binding)51 motif just N-terminal to a PDZ domain. This PDZ domain interacts with other proteins such as PALS119 and PAR3,52 presumably at the classic b2/a2-binding site. In the case of PALS1, this interaction is positively regulated by the Rho GTPase Cdc42-GTP through an association with the semi-CRIB domain of Par6.19 Importantly, an intact PDZ domain is required for this interaction.52 Although, this establishes yet another surprising role for the PDZ domain scaffold, the mechanism(s) by which Cdc42 exerts its regulation of the Par6 PDZ domain– PALS1 interaction remains unclear. Intriguingly, the two distal surfaces reported here are proximal to residues involved in Cdc42-binding of the Par6 semi-CRIB motif (see Figure 5C for specific interactions).18 Perturbations at or near these sites might influence binding at the peptide site by a mechanism that includes changes in fast timescale (ps – ns) dynamics in response to Cdc42binding, causing an allosteric response. The examples of GRIP1 and Par6 PDZ domains appear to be consistent with the surface linkages demonstrated here through perturbation of dynamics. It remains to be seen how frequently such secondary functional surfaces are utilized in other PDZ domains. It is possible, however, that such connectivity to distal surfaces may simply be intrinsic to the PDZ fold, and that this connectivity is exploited in only some cases. Further analysis of PDZ domains will provide additional insights into the role of dynamics in PDZ function and establish the extent of conservation of the dynamic pattern observed here.

Conclusions Allosteric communication is a fundamental process by which proteins propagate signals from one site to affect functionally important sites located

Dynamics of hPTP1E PDZ2

distally. Understanding the fundamental basis of this process remains a challenge. The qualitative correlation between thermodynamic residue– residue couplings and dynamic coupling described here suggests that dynamics may provide a mechanism for the transmission of allosteric signals throughout a protein. Alternatively, changes in protein dynamics may simply be a consequence of the transmission of this signal. Further studies are required to address this complicated issue.

Materials and Methods Cloning, expression and purification of hPTP1E PDZ2 PTP1E PDZ2 (1361-1456) was cloned from a human brain cDNA library (a gift from D. Siderovski, UNC Pharmacology) by use of the polymerase chain reaction and oligonucleotide primers designed from the DNA sequence (Genebank No. XM_172831).53 The cloned fragment was ligated into the pET 21a (Novagen) bacterial expression vector using Nde I and Bam HI restriction sites. A stop codon was engineered into the 30 primer, prior to the Bam HI site, so that a 96 amino acid residue fragment containing PDZ2 would be expressed. All cloning reactions were verified by automated sequencing (UNC sequencing facility). Over-expression of the PDZ2 protein was achieved by growing Escherichia coli strain BL21(DE3) harboring the expression plasmid in minimal media in the presence of ampicillin. Random, fractionally labeled 2H-methyl samples were produced using minimal media containing 15 NH4Cl, D -glucose (U-13C6-99%), and 60% 2H2O. Typically, 50 ml cultures of bacteria were grown overnight and used to inoculate one liter of culture media. The bacterial culture was incubated at 37 8C with vigorous shaking until an A600 nm of , 0.6– 0.8. Protein expression was induced with 1 mM IPTG for an additional incubation period of four to six hours at 37 8C. Cells were harvested by centrifugation and frozen at 220 8C until purification. Frozen pelleted cells were resuspended in buffer A (50 mM sodium acetate, pH 5.0) that included 10 mM EDTA, lyzed by sonication, and centrifuged. PDZ2 protein was purified using cation exchange chromatography (Pharmacia SP-Sepharose fast flow) in buffer A and eluted with 500 mM NaCl in buffer A. Elution from a G-50 gel filtration column in 50 mM phosphate, 150 mM NaCl (pH 6.8) resulted in . 95% purity, based on SDS-PAGE analysis. Typical yields were , 60 mg per liter of culture. The expected mass of the purified protein was confirmed by mass spectrometry analysis (UNC Proteomics Core). The RA-GEF2 peptide (Acetyl-ENEQVSAV-COOH) was synthesized using standard FMOC chemistry (UNC Microprotein Sequencing & Peptide Synthesis Facility) and HPLC purified to . 90% homogeneity and supplied as a lyophilized powder. The identity and purity of the peptide was confirmed by mass spectrometry. The PDZ2 – RA-GEF2 complex was formed by adding small amounts of concentrated peptide (,5 mM) to PDZ2 until saturation (the final stoichiometry was 1:1.25 (PDZ2– RA-GEF2)). The complex was lyophilized and resuspended in 90% H2O/10% 2H2O prior to NMR analysis. Protein NMR samples were generally , 2 mM in

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Dynamics of hPTP1E PDZ2

buffer containing 10% 2H2O, 50 mM phosphate, 150 mM NaCl at pH 6.8. NMR spectroscopy All NMR experiments were carried out at 25 8C (calibrated with methanol) on Varian Unity Inova spectrometers equipped with 1H/15N/13C probes and z-axis pulsed-field gradients. Previously published backbone spectral assignments were confirmed for the free24,54 and peptide-bound PDZ2. Side-chain methyl assignments were obtained using standard triple-resonance assignment experiments. Sterospecific assignments were achieved using a 10% 13C-PDZ2 sample55 in combination with the use of constant-time 13C-HSQC experiments.56 All NMR data were processed using NMRPipe 2.1 and analyzed using NMRView 5.04 compiled for Linux worksations. Standard backbone (15N) relaxation experiments,29 with minor modifications,49,57 were used to collect 15N T1 ; T1r ; and {1H}-15N NOE data at 500 and 600 MHz for free PDZ2 and the PDZ2 –RA-GEF2 complex (using 15Nlabeled PDZ2). T1 and T1r relaxation was typically sampled at eight to nine time points with three duplicate relaxation times per experiment. The 15N spin-lock employed in the T1r experiments was , 2.5 kHz. T1 relaxation times ranged from 0.035 second – 1.00 second for PDZ2 and PDZ2 – RA-GEF2. T1r relaxation times ranged from 5 – 136 ms. Two experiments comprising the {1H}– 15N NOE were acquired in an interleaved fashion, using a total recycle delay of ,5 seconds and 1H irradiation period of 4.5 seconds. Two-dimensional correlation spectra from each relaxation time point were processed into 2048 £ 512 matrices. Maximum peak intensities were used to generate relaxation decays for each resolved amide peak. Longitudinal and transverse relaxation decays were best-fitted to single exponentials using in-house routines. Transverse relaxation rates were corrected for resonance offset effects.58 Side-chain 2H-methyl relaxation experiments for CH2D isotopomers were collected for samples containing random fractional 2H-labeling. Three multi-coherence relaxation experiments (IzCz, IzCzDz, and IzCzDy) were collected at 500 MHz and 600 MHz.28 Typically, eight to nine relaxation time points were collected for both the free PDZ2 and PDZ2 – RA-GEF2 complex with an additional two to three duplicate points. Relaxation times for each experiment had the following ranges: IzCz, 10 – 80 ms; IzCzDz, 2 – 80 ms and IzCzDy, 1 – 30 ms. The details of data acquisition were essentially as published.28,37 The 13C carrier was placed at 18 ppm and 84 complex indirect carbon points were acquired using a 13C sweep width of ,23 ppm. The data were processed into 2048 £ 256 matrices for quantification of peak intensities. All decays were best-fitted to single exponentials. Relaxation analysis Initial studies for the free protein were carried out at 3.4 mM concentration. Control T1r experiments as a function of protein concentration verified that dynamics parameters were not concentration-dependent from 1.7 mM to 3.4 mM. All subsequent relaxation experiments were carried out at a protein concentration of , 2 mM. Prior to full analysis of 15N relaxation data, backbone amides located in flexible regions were identified and excluded for the determination of the overall rotational correlation time, tm : This process yielded 62 amides in the free state

and 67 amides in the complex whose NOE values were greater than 0.65 or whose T2 was statistically within the average.38 Rotational tumbling anisotropy was determined to be small ðDk =D’ ¼ 1:08 for free PDZ2; Dk =D’ ¼ 1:05 for PDZ2 – RA-GEF2) using the local Di approach implemented in the program Quadric Diffusion59 (Dr Art Palmer, Columbia University). Consequently, an isotropic tumbling model was used for subsequent analyses. Global fitting of data yielded an isotropic tm of 7.0 and 6.9 for the free PDZ2 and PDZ2– RA-GEF2 complex, respectively. This result agrees well with the determined tm (6.7 ns) for the mouse homologue obtained at similar conditions (25 8C and 1 mM protein concentration).24 Backbone dynamical parameters were fitted to the five standard motional models using ModelFree 4.1 (Dr Art Palmer, Columbia University) and the model selection criteria of Mandel et al.39 In all, 72/92 and 75/92 non-Pro NHs were analyzed for free and RA-GEF2 bound PDZ2. Side-chain methyl dynamical parameters were derived from S2 and te fits of the data as described.28,37 The final analysis yielded S2axis and te parameters for 49/62 methyl groups for the free PDZ2 and 47/62 methyl groups for the peptide complex.

Acknowledgements The authors thank members of the Lee laboratory, Drs Brian Volkman and Gary Pielak for helpful discussions and comments on the manuscript. E.J.F. is supported by an NSF Minority Fellowship.

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Edited by A. G. Palmer III (Received 28 July 2003; received in revised form 27 October 2003; accepted 6 November 2003)