The SrrAB two-component system regulates Staphylococcus aureus pathogenicity through redox sensitive cysteines.

Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. Here, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis.

Therapeutic Inhibition of Staphylococcus aureus ArlRS Two-Component Regulatory System Blocks Virulence.

Staphylococcus aureus is a common cause of severe infections, and its widespread antibiotic resistance necessitates search for alternative therapies, such as inhibition of virulence. As S. aureus produces multiple individual virulence factors, inhibition of an entire regulatory system might provide better effects than targeting each virulence factor separately. Herein, we describe two novel inhibitors of S. aureus two-component regulatory system ArlRS: 3,4'-dimethoxyflavone and homopterocarpin. Unlike other putative ArlRS inhibitors previously identified, these two compounds were effective and specific. In vitro kinase assays indicated that 3,4'-dimethoxyflavone directly inhibits ArlS autophosphorylation, while homopterocarpin did not exhibit such effect, suggesting that two inhibitors work through distinct mechanisms. Application of the inhibitors to methicillin-resistant S. aureus (MRSA) in vitro blocked ArlRS signaling, inducing an abnormal gene expression pattern that was reflected in changes at the protein level, enhanced sensitivity to oxacillin, and led to the loss of numerous cellular virulence traits, including the ability to clump, adhere to host ligands, and evade innate immunity. The pleiotropic antivirulence effect of inhibiting a single regulatory system resulted in a marked therapeutic potential, demonstrated by the ability of inhibitors to decrease severity of MRSA infection in mice. Altogether, this study demonstrated the feasibility of ArlRS inhibition as anti-S. aureus treatment, and identified new lead compounds for therapeutic development.

The Staphylococcus aureus ArlRS two-component system regulates virulence factor expression through MgrA.

The Gram-positive bacterium, Staphylococcus aureus, is a versatile pathogen that can sense and adapt to a wide variety of environments within the human host, in part through its 16 two-component regulatory systems. The ArlRS two-component system has been shown to affect many cellular processes in S. aureus, including autolysis, biofilm formation, capsule synthesis and virulence. Yet the molecular details of this regulation remained largely unknown. We used RNA sequencing to identify the ArlRS regulon, and found 70% overlap with that of the global regulator MgrA. These genes included cell wall-anchored adhesins (ebh, sdrD), polysaccharide and capsule synthesis genes, cell wall remodeling genes (lytN, ddh), the urease operon, genes involved in metal transport (feoA, mntH, sirA), anaerobic metabolism genes (adhE, pflA, nrdDG) and a large number of virulence factors (lukSF, lukAB, nuc, gehB, norB, chs, scn and esxA). We show that ArlR directly activates expression of mgrA and identify a probable ArlR-binding site (TTTTCTCAT-N4 -TTTTAATAA). A highly similar sequence is also found in the spx P2 promoter, which was recently shown to be regulated by ArlRS. We also demonstrate that ArlS has kinase activity toward ArlR in vitro, although it has slower kinetics than other similar histidine kinases.

Conformational Dynamics and Cooperativity Drive the Specificity of a Protein-Ligand Interaction.

Molecular recognition is critical for the fidelity of signal transduction in biology. Conversely, the disruption of protein-protein interactions can lead to disease. Thus, comprehension of the molecular determinants of specificity is essential for understanding normal biological signaling processes and for the development of precise therapeutics. Although high-resolution structures have provided atomic details of molecular interactions, much less is known about the influence of cooperativity and conformational dynamics. Here, we used the Tiam2 PSD-95/Dlg/ZO-1 (PDZ) domain and a quadruple mutant (QM), engineered by swapping the identity of four residues important for specificity in the Tiam1 PDZ into the Tiam2 PDZ domain, as a model system to investigate the role of cooperativity and dynamics in PDZ ligand specificity. Surprisingly, equilibrium binding experiments found that the ligand specificity of the Tiam2 QM was switched to that of the Tiam1 PDZ. NMR-based studies indicated that Tiam2 QM PDZ, but not other mutants, had extensive microsecond to millisecond motions distributed throughout the entire domain suggesting structural cooperativity between the mutated residues. Thermodynamic analyses revealed energetic cooperativity between residues in distinct specificity subpockets that was dependent upon the identity of the ligand, indicating a context-dependent binding mechanism. Finally, isothermal titration calorimetry experiments showed distinct entropic signatures along the mutational trajectory from the Tiam2 wild-type to the QM PDZ domain. Collectively, our studies provide unique insights into how structure, conformational dynamics, and thermodynamics combine to modulate ligand-binding specificity and have implications for the evolution, regulation, and design of protein-ligand interactions.

Accurate PDZ/Peptide Binding Specificity with Additive and Polarizable Free Energy Simulations.

PDZ domains contain 80-100 amino acids and bind short C-terminal sequences of target proteins. Their specificity is essential for cellular signaling pathways. We studied the binding of the Tiam1 PDZ domain to peptides derived from the C-termini of its Syndecan-1 and Caspr4 targets. We used free energy perturbation (FEP) to characterize the binding energetics of one wild-type and 17 mutant complexes by simulating 21 alchemical transformations between pairs of complexes. Thirteen complexes had known experimental affinities. FEP is a powerful tool to understand protein/ligand binding. It depends, however, on the accuracy of molecular dynamics force fields and conformational sampling. Both aspects require continued testing, especially for ionic mutations. For six mutations that did not modify the net charge, we obtained excellent agreement with experiment using the additive, AMBER ff99SB force field, with a root mean square deviation (RMSD) of 0.37 kcal/mol. For six ionic mutations that modified the net charge, agreement was also good, with one large error (3 kcal/mol) and an RMSD of 0.9 kcal/mol for the other five. The large error arose from the overstabilization of a protein/peptide salt bridge by the additive force field. Four of the ionic mutations were also simulated with the polarizable Drude force field, which represents the first test of this force field for protein/ligand binding free energy changes. The large error was eliminated and the RMS error for the four mutations was reduced from 1.8 to 1.2 kcal/mol. The overall accuracy of FEP indicates it can be used to understand PDZ/peptide binding. Importantly, our results show that for ionic mutations in buried regions, electronic polarization plays a significant role.

The Tiam1 guanine nucleotide exchange factor is auto-inhibited by its pleckstrin homology coiled-coil extension domain.

T-cell lymphoma invasion and metastasis 1 (Tiam1) is a Dbl-family guanine nucleotide exchange factor (GEF) that specifically activates the Rho-family GTPase Rac1 in response to upstream signals, thereby regulating cellular processes including cell adhesion and migration. Tiam1 contains multiple domains, including an N-terminal pleckstrin homology coiled-coiled extension (PHn-CC-Ex) and catalytic Dbl homology and C-terminal pleckstrin homology (DH-PHc) domain. Previous studies indicate that larger fragments of Tiam1, such as the region encompassing the N-terminal to C-terminal pleckstrin homology domains (PHn-PHc), are auto-inhibited. However, the domains in this region responsible for inhibition remain unknown. Here, we show that the PHn-CC-Ex domain inhibits Tiam1 GEF activity by directly interacting with the catalytic DH-PHc domain, preventing Rac1 binding and activation. Enzyme kinetics experiments suggested that Tiam1 is auto-inhibited through occlusion of the catalytic site rather than by allostery. Small angle X-ray scattering and ensemble modeling yielded models of the PHn-PHc fragment that indicate it is in equilibrium between "open" and "closed" conformational states. Finally, single-molecule experiments support a model in which conformational sampling between the open and closed states of Tiam1 contributes to Rac1 dissociation. Our results highlight the role of the PHn-CC-Ex domain in Tiam1 GEF regulation and suggest a combinatorial model for GEF inhibition and activation of the Rac1 signaling pathway.

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-ß5-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.

Assessing the functional roles of coevolving PHD finger residues.

Although in silico folding based on coevolving residue constraints in the deep-learning era has transformed protein structure prediction, the contributions of coevolving residues to protein folding, stability, and other functions in physical contexts remain to be clarified and experimentally validated. Herein, the PHD finger module, a well-known histone reader with distinct subtypes containing subtype-specific coevolving residues, was used as a model to experimentally assess the contributions of coevolving residues and to clarify their specific roles. The results of the assessment, including proteolysis and thermal unfolding of wildtype and mutant proteins, suggested that coevolving residues have varying contributions, despite their large in silico constraints. Residue positions with large constraints were found to contribute to stability in one subtype but not others. Computational sequence design and generative model-based energy estimates of individual structures were also implemented to complement the experimental assessment. Sequence design and energy estimates distinguish coevolving residues that contribute to folding from those that do not. The results of proteolytic analysis of mutations at positions contributing to folding were consistent with those suggested by sequence design and energy estimation. Thus, we report a comprehensive assessment of the contributions of coevolving residues, as well as a strategy based on a combination of approaches that should enable detailed understanding of the residue contributions in other large protein families.

High-resolution structure of the Tiam1 PHn-CC-Ex domain.

The T-lymphoma and metastasis gene 1 (TIAM1) encodes a guanine nucleotide-exchange factor protein (Tiam1) that is specific for the Rho-family GTPase Rac1 and is important for cell polarity, migration and adhesion. Tiam1 is a large multi-domain protein that contains several protein-protein binding domains that are important for regulating cellular function. The PHn-CC-Ex domain is critical for plasma-membrane association and interactions with protein-scaffold proteins (e.g. Par3b, spinophilin, IRSp53 and JIP2) that direct Tiam1-Rac1 signaling specificity. It was determined that the coiled-coil domain of Par3b binds the PHn-CC-Ex domain with a dissociation constant of ≈ 30 µM. Moreover, the structures of two variants of the Tiam1 PHn-CC-Ex domain were solved at resolutions of 1.98 and 2.15 Å, respectively. The structures indicate that the PHn, CC and Ex regions form independent subdomains that together provide an integrated platform for binding partner proteins. Small-angle X-ray scattering (SAXS) data indicate that the Tiam1 PHn-CC-Ex domain is monomeric in solution and that the solution and crystal structures are very similar. Together, these data provide the foundation necessary to elucidate the structural mechanism of the PHn-CC-Ex/scaffold interactions that are critical for Tiam1-Rac1 signaling specificity.

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 protein's 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 (alpha3) that packs in contiguous fashion against the globular domain. Although alpha3 lies outside the active site and does not make direct contact with C-terminal peptide ligand, removal of alpha3 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 alpha3, picosecond-nanosecond side-chain dynamics are enhanced throughout the domain, as determined by (2)H methyl NMR relaxation. Thus, the distal modulation of binding function appears to occur via a delocalized conformational entropy mechanism. Without removal of alpha3 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 alpha3, 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.

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.

A Fluorescence-Based Assay to Determine PDZ-Ligand Binding Thermodynamics.

Postsynaptic density-95, disks-large, and zonula occludens-1 (PDZ) domain interactions with cognate linear binding motifs (i.e., PDZ-binding motifs or PBMs) are important for many biological processes and can be pathological when disrupted. There are hundreds of PDZ-PBM interactions reported but few have been quantitatively determined. Moreover, PDZ-PBM interactions have been identified as potential therapeutic targets. To thoroughly understand PDZ-PBM binding energetics and their specificity, we have developed a sensitive and quantitative equilibrium binding assay. Here, we describe a protocol for determining PDZ-PBM binding energetics using fluorescence anisotropy-based methodology.

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.

A physics-based energy function allows the computational redesign of a PDZ domain.

Computational protein design (CPD) can address the inverse folding problem, exploring a large space of sequences and selecting ones predicted to fold. CPD was used previously to redesign several proteins, employing a knowledge-based energy function for both the folded and unfolded states. We show that a PDZ domain can be entirely redesigned using a "physics-based" energy for the folded state and a knowledge-based energy for the unfolded state. Thousands of sequences were generated by Monte Carlo simulation. Three were chosen for experimental testing, based on their low energies and several empirical criteria. All three could be overexpressed and had native-like circular dichroism spectra and 1D-NMR spectra typical of folded structures. Two had upshifted thermal denaturation curves when a peptide ligand was present, indicating binding and suggesting folding to a correct, PDZ structure. Evidently, the physical principles that govern folded proteins, with a dash of empirical post-filtering, can allow successful whole-protein redesign.

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 protein's 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.

Emerging Themes in PDZ Domain Signaling: Structure, Function, and Inhibition.

Post-synaptic density-95, disks-large and zonula occludens-1 (PDZ) domains are small globular protein-protein interaction domains widely conserved from yeast to humans. They are composed of ∼90 amino acids and form a classical two α-helical/six ß-strand structure. The prototypical ligand is the C-terminus of partner proteins; however, they also bind internal peptide sequences. Recent findings indicate that PDZ domains also bind phosphatidylinositides and cholesterol. Through their ligand interactions, PDZ domain proteins are critical for cellular trafficking and the surface retention of various ion channels. In addition, PDZ proteins are essential for neuronal signaling, memory, and learning. PDZ proteins also contribute to cytoskeletal dynamics by mediating interactions critical for maintaining cell-cell junctions, cell polarity, and cell migration. Given their important biological roles, it is not surprising that their dysfunction can lead to multiple disease states. As such, PDZ domain-containing proteins have emerged as potential targets for the development of small molecular inhibitors as therapeutic agents. Recent data suggest that the critical binding function of PDZ domains in cell signaling is more than just glue, and their binding function can be regulated by phosphorylation or allosterically by other binding partners. These studies also provide a wealth of structural and biophysical data that are beginning to reveal the physical features that endow this small modular domain with a central role in cell signaling.

SGEF forms a complex with Scribble and Dlg1 and regulates epithelial junctions and contractility.

The canonical Scribble polarity complex is implicated in regulation of epithelial junctions and apical polarity. Here, we show that SGEF, a RhoG-specific GEF, forms a ternary complex with Scribble and Dlg1, two members of the Scribble complex. SGEF targets to apical junctions in a Scribble-dependent fashion and functions in the regulation of actomyosin-based contractility and barrier function at tight junctions as well as E-cadherin-mediated formation of adherens junctions. Surprisingly, SGEF does not control the establishment of polarity. However, in 3D cysts, SGEF regulates the formation of a single open lumen. Interestingly, SGEF's nucleotide exchange activity regulates the formation and maintenance of adherens junctions, and in cysts the number of lumens formed, whereas SGEF's scaffolding activity is critical for regulation of actomyosin contractility and lumen opening. We propose that SGEF plays a key role in coordinating junctional assembly and actomyosin contractility by bringing together Scribble and Dlg1 and targeting RhoG activation to cell-cell junctions.

Evaluation of energetic and dynamic coupling networks in a PDZ domain protein.

A number of computational and experimental studies have identified intramolecular communication "pathways" or "networks" important for transmitting allostery. Here, we have used mutagenesis and NMR relaxation methods to investigate the scope and nature of the communication networks found in the second post-synaptic density-95/discs large/zonula occludens-1 (PDZ) domain of the human protein tyrosine phosphatase 1E protein (hPTP1E) (PDZ2). It was found that most mutations do not have a significant energetic contribution to peptide ligand binding. Three mutants that showed significant changes in binding also displayed context-dependent dynamic effects. Both a mutation at a partially exposed site (H71Y) and a buried core position (I35V) had a limited response in side-chain (2)H-based dynamics when compared to wild-type PDZ2. In contrast, a change at a second core position (I20F) that had previously been shown to be part of an energetic and dynamic network, resulted in extensive changes in side-chain dynamics. This response is reminiscent to that seen previously upon peptide ligand binding. These results shed light on the nature of the PDZ2 dynamic network and suggest that position 20 in PDZ2 acts as a "hub" that is energetically and dynamically critical for transmitting changes in dynamics throughout the PDZ domain.

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.

Computational Design of the Tiam1 PDZ Domain and Its Ligand Binding.

PDZ domains direct protein-protein interactions and serve as models for protein design. Here, we optimized a protein design energy function for the Tiam1 and Cask PDZ domains that combines a molecular mechanics energy, Generalized Born solvent, and an empirical unfolded state model. Designed sequences were recognized as PDZ domains by the Superfamily fold recognition tool and had similarity scores comparable to natural PDZ sequences. The optimized model was used to redesign the two PDZ domains, by gradually varying the chemical potential of hydrophobic amino acids; the tendency of each position to lose or gain a hydrophobic character represents a novel hydrophobicity index. We also redesigned four positions in the Tiam1 PDZ domain involved in peptide binding specificity. The calculated affinity differences between designed variants reproduced experimental data and suggest substitutions with altered specificities.