Ernesto J. Fuentes

Hidden dynamic allostery in a PDZ domain

Structure–function relationships in proteins are predicated on the spatial proximity of noncovalently interacting groups of atoms. Thus, structural elements located away from a proteins active site are typically presumed to serve a stabilizing or scaffolding role for the larger structure. Here we report a functional role for a distal structural element in a PDZ domain, even though it is not required to maintain PDZ structure. The third PDZ domain from PSD-95/SAP90 (PDZ3) has an unusual additional third alpha helix (α3) that packs in contiguous fashion against the globular domain. Although α3 lies outside the active site and does not make direct contact with C-terminal peptide ligand, removal of α3 reduces ligand affinity by 21-fold. Further investigation revealed that the difference in binding free energies between the full-length and truncated constructs is predominantly entropic in nature and that without α3, picosecond-nanosecond side-chain dynamics are enhanced throughout the domain, as determined by 2H methyl NMR relaxation. Thus, the distal modulation of binding function appears to occur via a delocalized conformational entropy mechanism. Without removal of α3 and characterization of side-chain dynamics, this dynamic allostery would have gone unnoticed. Moreover, what appeared at first to be an artificial modification of PDZ3 has been corroborated by experimentally verified phosphorylation of α3, revealing a tangible biological mechanism for this novel regulatory scheme. This hidden dynamic allostery raises the possibility of as-yet unidentified or untapped allosteric regulation in this PDZ domain and is a very clear example of function arising from dynamics rather than from structure.

A novel pathway spatiotemporally activates Rac1 and redox signaling in response to fluid shear stress

The GEF Tiam1 acts as a novel molecular link to the VE-cadherin–p67phox–Par3 polarity complex, leading to localized activation of Rac1 and NADPH oxidase in response to fluid flow.

Conservation of Side-Chain Dynamics Within a Protein Family

The question of protein dynamics and its relevance to function is currently a topic of great interest. Proteins are particularly dynamic at the side-chain level on the time scale of picoseconds to nanoseconds. Here, we present a comparison of NMR-monitored side-chain motion between three PDZ domains of approximately 30% sequence identity and show that the side-chain dynamics display nontrivial conservation. Methyl (2)H relaxation was carried out to determine side-chain order parameters (S(2)), which were found to be more similar than naively expected from sequence, local packing, or a combination of the two. Thus, the dynamics of a rather distant homologue appears to be an excellent predictor of a proteins side-chain dynamics and, on average, better than current structure-based methods. Fast side-chain dynamics therefore display a high level of organization associated with global fold. Beyond simple conservation, the analysis herein suggests that the pattern of side-chain flexibility has significant contributions from nonlocal elements of the PDZ fold, such as correlated motions, and that the conserved dynamics may directly support function.

Specificity Residues Determine Binding Affinity for Two-Component Signal Transduction Systems

ABSTRACT Two-component systems (TCS) comprise histidine kinases and their cognate response regulators and allow bacteria to sense and respond to a wide variety of signals. Histidine kinases (HKs) phosphorylate and dephosphorylate their cognate response regulators (RRs) in response to stimuli. In general, these reactions appear to be highly specific and require an appropriate association between the HK and RR proteins. The Myxococcus xanthus genome encodes one of the largest repertoires of signaling proteins in bacteria (685 open reading frames [ORFs]), including at least 127 HKs and at least 143 RRs. Of these, 27 are bona fide NtrC-family response regulators, 21 of which are encoded adjacent to their predicted cognate kinases. Using system-wide profiling methods, we determined that the HK-NtrC RR pairs display a kinetic preference during both phosphotransfer and phosphatase functions, thereby defining cognate signaling systems in M. xanthus. Isothermal titration calorimetry measurements indicated that cognate HK-RR pairs interact with dissociation constants (Kd) of approximately 1 µM, while noncognate pairs had no measurable binding. Lastly, a chimera generated between the histidine kinase, CrdS, and HK1190 revealed that residues conferring phosphotransfer and phosphatase specificity dictate binding affinity, thereby establishing discrete protein-protein interactions which prevent cross talk. The data indicate that binding affinity is a critical parameter governing system-wide signaling fidelity for bacterial signal transduction proteins. IMPORTANCE Using in vitro phosphotransfer and phosphatase profiling assays and isothermal titration calorimetry, we have taken a system-wide approach to demonstrate specificity for a family of two-component signaling proteins in Myxococcus xanthus. Our results demonstrate that previously identified specificity residues dictate binding affinity and that phosphatase specificity follows phosphotransfer specificity for cognate HK-RR pairs. The data indicate that preferential binding affinity is the basis for signaling fidelity in bacterial two-component systems. Using in vitro phosphotransfer and phosphatase profiling assays and isothermal titration calorimetry, we have taken a system-wide approach to demonstrate specificity for a family of two-component signaling proteins in Myxococcus xanthus. Our results demonstrate that previously identified specificity residues dictate binding affinity and that phosphatase specificity follows phosphotransfer specificity for cognate HK-RR pairs. The data indicate that preferential binding affinity is the basis for signaling fidelity in bacterial two-component systems.

Distinct Ligand Specificity of the Tiam1 and Tiam2 PDZ Domains

Guanine nucleotide exchange factor proteins of the Tiam family are activators of the Rho GTPase Rac1 and critical for cell morphology, adhesion, migration, and polarity. These proteins are modular and contain a variety of interaction domains, including a single post-synaptic density-95/discs large/zonula occludens-1 (PDZ) domain. Previous studies suggest that the specificities of the Tiam1 and Tiam2 PDZ domains are distinct. Here, we sought to conclusively define these specificities and determine their molecular origin. Using a combinatorial peptide library, we identified a consensus binding sequence for each PDZ domain. Analysis of these consensus sequences and binding assays with peptides derived from native proteins indicated that these two PDZ domains have overlapping but distinct specificities. We also identified residues in two regions (S(0) and S(-2) pockets) of the Tiam1 PDZ domain that are important determinants of ligand specificity. Site-directed mutagenesis of four nonconserved residues in these two regions along with peptide binding analyses confirmed that these residues are crucial for ligand affinity and specificity. Furthermore, double mutant cycle analysis of each region revealed energetic couplings that were dependent on the ligand being investigated. Remarkably, a Tiam1 PDZ domain quadruple mutant had the same specificity as the Tiam2 PDZ domain. Finally, analysis of Tiam family PDZ domain sequences indicated that the PDZ domains segregate into four distinct families based on the residues studied here. Collectively, our data suggest that Tiam family proteins have highly evolved PDZ domain-ligand interfaces with distinct specificities and that they have disparate PDZ domain-dependent biological functions.

The structure of the Tiam1 PDZ domain/ phospho-syndecan1 complex reveals a ligand conformation that modulates protein dynamics.

PDZ (PSD-95/Dlg/ZO-1) domains are protein-protein interaction modules often regulated by ligand phosphorylation. Here, we investigated the specificity, structure, and dynamics of Tiam1 PDZ domain/ligand interactions. We show that the PDZ domain specifically binds syndecan1 (SDC1), phosphorylated SDC1 (pSDC1), and SDC3 but not other syndecan isoforms. The crystal structure of the PDZ/SDC1 complex indicates that syndecan affinity is derived from amino acids beyond the four C-terminal residues. Remarkably, the crystal structure of the PDZ/pSDC1 complex reveals a binding pocket that accommodates the phosphoryl group. Methyl relaxation experiments of PDZ/SCD1 and PDZ/pSDC1 complexes reveal that PDZ-phosphoryl interactions dampen dynamic motions in a distal region of the PDZ domain by decoupling them from the ligand-binding site. Our data are consistent with a selection model by which specificity and phosphorylation regulate PDZ/syndecan interactions and signaling events. Importantly, our relaxation data demonstrate that PDZ/phospho-ligand interactions regulate protein dynamics and their coupling to distal sites.

In silico prediction of the 3D structure of trimeric asialoglycoprotein receptor bound to triantennary oligosaccharide.

In this study, we present a general-purpose methodology for deriving the three-dimensional (3D) arrangement of multivalent transmembrane complexes in the presence of their ligands. Specifically, we predict the most likely families of structures of the experimentally intractable trimeric asialoglycoprotein receptor (ASGP-R), which consists of human hepatic subunits (two subunits of H1 and one subunit of H2), bound to a triantennary oligosaccharide (TA). Because of the complex nature of this multivalent type-II transmembrane hetero-oligomeric receptor, structural studies have to date been unable to provide the 3D arrangement of these subunits. Our approach is based on using the three-pronged ligand of ASGP-R as a computational probe to derive the 3D conformation of the complex and then using this information to predict the relative arrangement of the protein subunits on the cell surface. Because of interprotein subunit clashes, only a few families of TA conformers are compatible with the trimeric structure of ASGP-R. We find that TA displays significant flexibility, matching that detected previously in FRET experiments, and that the predicted complexes derived from the viable TA structures are asymmetric. Significant variation exists with respect to TA presentation to the receptor complex. In summary, this study provides detailed information about TA-ASGP-R interactions and the symmetry of the complex.

Chemosensory Regulation of a HEAT-Repeat Protein Couples Aggregation and Sporulation in Myxococcus xanthus

Chemosensory systems are complex, highly modified two-component systems (TCS) used by bacteria to control various biological functions ranging from motility to sporulation. Chemosensory systems and TCS both modulate phosphorelays comprised of histidine kinases and response regulators, some of which are single-domain response regulators (SD-RRs) such as CheY. In this study, we have identified and characterized the Che7 chemosensory system of Myxococcus xanthus, a common soil bacterium which displays multicellular development in response to stress. Both genetic and biochemical analyses indicate that the Che7 system regulates development via a direct interaction between the SD-RR CheY7 and a HEAT repeat domain-containing protein, Cpc7. Phosphorylation of the SD-RR affects the interaction with its target, and residues within the α4-β5-α5 fold of the REC domain govern this interaction. The identification of the Cpc7 interaction with CheY7 extends the diversity of known targets for SD-RRs in biological systems.

FBG1 is a promiscuous ubiquitin ligase that sequesters APC2 and causes S-phase arrest

During cell proliferation, protein degradation is strictly regulated by the cell cycle and involves two complementary ubiquitin ligase complexes, the SCF (Skp, Cullin, F-box) and APC/C (Anaphase Promoting Complex/Cyclosome) ubiquitin ligases. SCF ligases are constitutively active and generally target only proteins after they have been selected for degradation, usually by phosphorylation. In contrast, APC/C complexes are themselves activated by phosphorylation and their substrates contain a targeting signal known as degron, a consensus amino acid sequence such as a D-Box. SCF complexes degrade proteins during the G1 phase. However, as DNA synthesis begins, the SCF complexes are degraded and APC/C complexes are activated. APC-2, a protein crucial to cell division, initiates anaphase by triggering the degradation of multiple proteins. This study explores an unexpected interaction between APC-2 and SCFFBG1. We found that FBG1 is a promiscuous ubiquitin ligase with many partners. Immunoprecipitation experiments demonstrate that FBG1 and APC2 interact directly. Mutagenesis-based experiments show that this interaction requires a D-Box found within the FBG1 F-box domain. Unexpectedly, we demonstrate that co-expression with FBG1 increases total APC2 levels. However, free APC2 is decreased, inhibiting cell proliferation. Finally, FACS analysis of cell populations expressing different forms of FBG1 demonstrate that this ubiquitin ligase induces S-phase arrest, illustrating the functional consequences of the interaction described. In summary, we have discovered a novel APC2 inhibitory activity of FBG1 independent from its function as ubiquitin ligase, providing the basis for future studies of FBG1 in aging and cancer.

A Simple PB/LIE Free Energy Function Accurately Predicts the Peptide Binding Specificity of the Tiam1 PDZ Domain

PDZ domains generally bind short amino acid sequences at the C-terminus of target proteins, and short peptides can be used as inhibitors or model ligands. Here, we used experimental binding assays and molecular dynamics simulations to characterize 51 complexes involving the Tiam1 PDZ domain and to test the performance of a semi-empirical free energy function. The free energy function combined a Poisson-Boltzmann (PB) continuum electrostatic term, a van der Waals interaction energy, and a surface area term. Each term was empirically weighted, giving a Linear Interaction Energy or “PB/LIE” free energy. The model yielded a mean unsigned deviation of 0.43 kcal/mol and a Pearson correlation of 0.64 between experimental and computed free energies, which was superior to a Null model that assumes all complexes have the same affinity. Analyses of the models support several experimental observations that indicate the orientation of the α2 helix is a critical determinant for peptide specificity. The models were also used to predict binding free energies for nine new variants, corresponding to point mutants of the Syndecan1 and Caspr4 peptides. The predictions did not reveal improved binding; however, they suggest that an unnatural amino acid could be used to increase protease resistance and peptide lifetimes in vivo. The overall performance of the model should allow its use in the design of new PDZ ligands in the future.