Α1 Helix Research Articles

Structural analysis of the recognition of the -35 promoter element by SigW from Bacillus subtilis.

Sigma factors are key proteins that mediate the recruitment of RNA polymerase to the promoter regions of genes, for the initiation of bacterial transcription. Multiple sigma factors in a bacterium selectively recognize their cognate promoter sequences, thereby inducing the expression of their own regulons. In this paper, we report the crystal structure of the σ4 domain of Bacillus subtilis SigW bound to the -35 promoter element. Purine-specific hydrogen bonds of the -35 promoter element with the recognition helix α9 of the σ4 domain occurs at three nucleotides of the consensus sequence (G-35, A-34, and G’-31 in G-35A-34A-33A-32C-31C-30T-29). The hydrogen bonds of the backbone with the α7 and α8 of the σ4 domain occurs at G’-30. These results elucidate the structural basis of the selective recognition of the promoter by SigW. In addition, comparison of SigW structures complexed with the -35 promoter element or with anti-sigma RsiW reveals that DNA recognition and anti-sigma factor binding of SigW are mutually exclusive.

Structures of kobuviral and siciniviral polymerases reveal conserved mechanism of picornaviral polymerase activation

RNA-dependent RNA polymerase 3Dpol is a key enzyme for the replication of picornaviruses. The viral genome is translated into a single polyprotein that is subsequently proteolytically processed into matured products. The 3Dpol enzyme arises from a stable 3CD precursor that has high proteolytic activity but no polymerase activity. Upon cleavage of the precursor the newly established N-terminus of 3Dpol is liberated and inserts itself into a pocket on the surface of the 3Dpol enzyme. The essential residue for this mechanism is the very first glycine that is conserved among almost all picornaviruses. However, kobuviruses and siciniviruses have a serine residue instead. Intrigued by this anomaly we sought to solve the crystal structure of these 3Dpol enzymes. The structures revealed a unique fold of the 3Dpol N-termini but the very first serine residues were inserted into a charged pocket in a similar manner as the glycine residue in other picornaviruses. These structures revealed a common underlying mechanism of 3Dpol activation that lies in activation of the α10 helix containing a key catalytical residue Asp238 that forms a hydrogen bond with the 2′ hydroxyl group of the incoming NTP nucleotide.

Structure of the primed state of the ATPase domain of chromatin remodeling factor ISWI bound to the nucleosome.

ATP-dependent chromatin remodeling factors of SWI/SNF2 family including ISWI, SNF2, CHD1 and INO80 subfamilies share a conserved but functionally non-interchangeable ATPase domain. Here we report cryo-electron microscopy (cryo-EM) structures of the nucleosome bound to an ISWI fragment with deletion of the AutoN and HSS regions in nucleotide-free conditions and the free nucleosome at ∼ 4 Å resolution. In the bound conformation, the ATPase domain interacts with the super helical location 2 (SHL 2) of the nucleosomal DNA, with the N-terminal tail of H4 and with the α1 helix of H3. Density for other regions of ISWI is not observed, presumably due to disorder. Comparison with the structure of the free nucleosome reveals that although the histone core remains largely unchanged, remodeler binding causes perturbations in the nucleosomal DNA resulting in a bulge near the SHL2 site. Overall, the structure of the nucleotide-free ISWI-nucleosome complex is similar to the corresponding regions of the recently reported ADP bound ISWI-nucleosome structures, which are significantly different from that observed for the ADP-BeFx bound structure. Our findings are relevant to the initial step of ISWI binding to the nucleosome and provide additional insights into the nucleosome remodeling process driven by ISWI.

Tyrosine 51 residue of the syndecan-2 extracellular domain is involved in the interaction with and activation of pro-matrix metalloproteinase-7

Although syndecan-2 is known to interact with the matrix metalloproteinase-7 (MMP-7), the details of their interaction were unknown. Our experiments with a series of syndecan-2 extracellular domain deletion mutants show that the interaction is mediated through an interaction of the extracellular domain of syndecan-2 (residues 41 to 60) with the α2 helix-loop-α3 helix in the pro-domain of MMP-7. NMR and molecular docking model show that Glu7 of the α1 helix, Glu32 of the α2 helix, and Gly48 and Ser52 of the α2 helix-loop-α3 helix of the MMP-7 pro-domain form the syndecan-2-binding pocket, which is occupied by the side chain of tyrosine residue 51 (Tyr51) of syndecan-2. Consistent with this notion, the expression of a syndecan-2 mutant in which Tyr51 was changed to Ala diminished the interaction between the syndecan-2 extracellular domain and the pro-domain of MMP-7. Furthermore, HT-29 colon adenocarcinoma cells expressing the interaction-defective mutant exhibited reductions in the cell-surface localization of MMP-7, the processing of pro-MMP-7 into active MMP-7, the MMP-7-mediated extracellular domain shedding of both syndecan-2 and E-cadherin, and syndecan-2-mediated anchorage-independent growth. Collectively, these data strongly suggest that Tyr51 of the syndecan-2 extracellular domain mediates its interaction with and activating processing of pro-MMP-7 and regulates MMP-7-dependent syndecan-2 functions.

Crystal structure of CagV, the Helicobacter pylori homologue of the T4SS protein VirB8.

The VirB/D type IV secretion system (T4SS) plays an essential role in materials transport between host cells and pathogenic Helicobacter pylori and is considered the major pathogenic mediator of H.pylori-associated gastric disease. VirB8, an inner membrane protein that interacts with many other proteins, is a crucial component for secretory function. Here, we present a crystal structure of the periplasmic domain of CagV, the VirB8 counterpart in the H.pylori Cag-T4SS. The structure reveals a fold similar to that of other VirB8 members except for the absence of the α5 helix, a discontinuous β1 strand, a larger angle between the α2 and α3 helices, a more hydrophobic surface groove, but exhibits a different dimer interface. Whether the dimerization occurs in solution was proved by mutagenesis, size-exclusion chromatography and cross-linking assays. Unlike the classical dimerization mode, the interface of the CagV dimer is principally formed by several hydrogen bonds, which indicates instability of dimerization. The structure here demonstrates the difference in dimerization among VirB8 homologues and indicates the considerable compositional and functional diversity of them in T4SS. DATABASE: Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 6IQT.

The Observation of Ligand-Binding-Relevant Open States of Fatty Acid Binding Protein by Molecular Dynamics Simulations and a Markov State Model.

As a member of the fatty acids transporter family, the heart fatty acid binding proteins (HFABPs) are responsible for many important biological activities. The binding mechanism of fatty acid with FABP is critical to the understanding of FABP functions. The uncovering of binding-relevant intermediate states and interactions would greatly increase our knowledge of the binding process. In this work, all-atom molecular dynamics (MD) simulations were performed to characterize the structural properties of nativelike intermediate states. Based on multiple 6 μs MD simulations and Markov state model (MSM) analysis, several “open” intermediate states were observed. The transition rates between these states and the native closed state are in good agreement with the experimental measurements, which indicates that these intermediate states are binding relevant. As a common property in the open states, the partially unfolded α2 helix generates a larger portal and provides the driving force to facilitate ligand binding. On the other side, there are two kinds of open states for the ligand-binding HFABP: one has the partially unfolded α2 helix, and the other has the looser β-barrel with disjointing βD-βE strands. Our results provide atomic-level descriptions of the binding-relevant intermediate states and could improve our understanding of the binding mechanism.

Mechanistic Insights into Specific G Protein Interactions with Adenosine Receptors.

Coupling between G-protein-coupled receptors (GPCRs) and the G proteins is a key step in cellular signaling. Despite extensive experimental and computational studies, the mechanism of specific GPCR-G protein coupling remains poorly understood. This has greatly hindered effective drug design of GPCRs that are primary targets of ∼1/3 of currently marketed drugs. Here, we have employed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method to decipher the mechanism of the GPCR-G protein interactions. Adenosine receptors (ARs) were used as model systems based on very recently determined cryo-EM structures of the A1AR and A2AAR coupled with the Gi and Gs proteins, respectively. Changing the Gi protein to the Gs led to increased fluctuations in the A1AR and agonist adenosine (ADO), while agonist 5'-N-ethylcarboxamidoadenosine (NECA) binding in the A2AAR could be still stabilized upon changing the Gs protein to the Gi. Free energy calculations identified one stable low-energy conformation for each of the A1AR-Gi and A2AAR-Gs complexes as in the cryo-EM structures, similarly for the A2AAR-Gi complex. In contrast, the ADO agonist and Gs protein sampled multiple conformations in the A1AR-Gs system. GaMD simulations thus indicated that the A1AR preferred to couple with the Gi protein to the Gs, while the A2AAR could couple with both the Gs and Gi proteins, being highly consistent with experimental findings of the ARs. More importantly, detailed analysis of the atomic simulations showed that the specific AR-G protein coupling resulted from remarkably complementary residue interactions at the protein interface, involving mainly the receptor transmembrane 6 helix and the Gα α5 helix and α4-β6 loop. In summary, the GaMD simulations have provided unprecedented insights into the dynamic mechanism of specific GPCR-G protein interactions at an atomistic level.

PEA-15 C-Terminal Tail Allosterically Modulates Death-Effector Domain Conformation and Facilitates Protein-Protein Interactions.

Phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) exerts its regulatory roles on several critical cellular pathways through protein–protein interactions depending on its phosphorylation states. It can either inhibit the extracellular signal-regulated kinase (ERK) activities when it is dephosphorylated or block the assembly of death-inducing signaling complex (DISC) and the subsequent activation of apoptotic initiator, caspase-8, when it is phosphorylated. Due to the important roles of PEA-15 in regulating these pathways that lead to opposite cellular outcomes (cell proliferation vs. cell death), we proposed a phosphostasis (phosphorylation homeostasis) model, in which the phosphorylation states of the protein are vigorously controlled and regulated to maintain a delicate balance. The phosphostasis gives rise to the protective cellular functions of PEA-15 to preserve optimum cellular conditions. In this article, using advanced multidimensional nuclear magnetic resonance (NMR) techniques combined with a novel chemical shift (CS)-Rosetta algorithm for de novo protein structural determination, we report a novel conformation of PEA-15 death-effector domain (DED) upon interacting with ERK2. This new conformation is modulated by the irregularly structured C-terminal tail when it first recognizes and binds to ERK2 at the d-peptide recruitment site (DRS) in an allosteric manner, and is facilitated by the rearrangement of the surface electrostatic and hydrogen-bonding interactions on the DED. In this ERK2-bound conformation, three of the six helices (α2, α3, and α4) comprising the DED reorient substantially in comparison to the free-form structure, exposing key residues on the other three helices that directly interact with ERK2 at the DEF-docking site (docking site for ERK, FxF) and the activation loop. Additionally, we provide evidence that the phosphorylation of the C-terminal tail leads to a distinct conformation of DED, allowing efficient interactions with Fas-associated death domain (FADD) protein at the DISC. Our results substantiate the allosteric regulatory roles of the C-terminal tail in modulating DED conformation and facilitating protein–protein interactions of PEA-15.

Shaft Function of Kinesin-1's α4 Helix in the Processive Movement.

Kinesin-1 motor is a molecular walking machine constructed with amino acids. The understanding of how those structural elements play their mechanical roles is the key to the understanding of kinesin-1 mechanism. Using molecular dynamics simulations, we investigate the role of a helix structure, α4 (also called switch-II helix), of kinesin-1's motor domain in its processive movement along microtubule. Through the analysis of the structure and the interactions between α4 and the surrounding residues in different nucleotide-binding states, we find that, mechanically, this helix functions as a shaft for kinesin-1's motor-domain rotation and, structurally, it is an amphipathic helix ensuring its shaft functioning. The hydrophobic side of α4 consists strictly of hydrophobic residues, making it behave like a lubricated surface in contact with the core β-sheet of kinesin-1's motor domain. The opposite hydrophilic side of α4 leans firmly against microtubule with charged residues locating at both ends to facilitate its positioning onto the intra-tubulin groove. The special structural feature of α4 makes for an effective reduction of the conformational work in kinesin-1's force generation process.

Monitoring site-specific conformational changes in real-time reveals a misfolding mechanism of the prion protein.

During pathological aggregation, proteins undergo remarkable conformational re-arrangements to anomalously assemble into a heterogeneous collection of misfolded multimers, ranging from soluble oligomers to insoluble amyloid fibrils. Inspired by fluorescence resonance energy transfer (FRET) measurements of protein folding, an experimental strategy to study site-specific misfolding kinetics during aggregation, by effectively suppressing contributions from inter-molecular FRET, is described. Specifically, the kinetics of conformational changes across different secondary and tertiary structural segments of the mouse prion protein (moPrP) were monitored independently, after the monomeric units transformed into large oligomers OL, which subsequently disaggregated reversibly into small oligomers OS at pH 4. The sequence segments spanning helices α2 and α3 underwent a compaction during the formation of OL and elongation into β-sheets during the formation of OS. The β1-α1-β2 and α2-α3 subdomains were separated, and the helix α1 was unfolded to varying extents in both OL and OS.

An In-Silico Sequence-Structure-Function Analysis of the N-Terminal Lobe in CT Group Bacterial ADP-Ribosyltransferase Toxins

The C3-like toxins are single-domain proteins that represent a minimal mono-ADP-ribosyl transferase (mART) enzyme with a simple model scaffold for the entire cholera toxin (CT)-group. These proteins possess a single (A-domain) that modifies Rho proteins. In contrast, C2-like toxins require a binding/translocation partner (B-component) for intoxication. These are A-only toxins that contain the E-x-E motif, modify G-actin, but are two-domains with a C-domain possessing enzymatic activity. The N-domain of the C2-like toxins is unstructured, and its function is currently unknown. A sequence-structure-function comparison was performed on the N-terminal region of the mART domain of the enzymatic component of the CT toxin group in the CATCH fold (3.90.210.10). Special consideration was given to the N-domain distal segment, the α-lobe (α1–α4), and its different roles in these toxin sub-groups. These results show that the role of the N-terminal α-lobe is to provide a suitable configuration (i) of the α2–α3 helices to feature the α3-motif that has a role in NAD+ substrate binding and possibly in the interaction with the protein target; (ii) the α3–α4 helices to provide the α3/4-loop with protein-protein interaction capability; and (iii) the α1-Ntail that features specialized motif(s) according to the toxin type (A-only or A-B toxins) exhibiting an effect on the catalytic activity via the ARTT-loop, with a role in the inter-domain stability, and with a function in the binding and/or translocation steps during the internalization process.

Structural mechanism for guanylate-binding proteins (GBPs) targeting by the Shigella E3 ligase IpaH9.8.

The guanylate-binding proteins (GBPs) belong to the dynamin superfamily of GTPases and function in cell-autonomous defense against intracellular pathogens. IpaH9.8, an E3 ligase from the pathogenic bacterium Shigella flexneri, ubiquitinates a subset of GBPs and leads to their proteasomal degradation. Here we report the structure of a C-terminally truncated GBP1 in complex with the IpaH9.8 Leucine-rich repeat (LRR) domain. IpaH9.8LRR engages the GTPase domain of GBP1, and differences in the Switch II and α3 helix regions render some GBPs such as GBP3 and GBP7 resistant to IpaH9.8. Comparisons with other IpaH structures uncover interaction hot spots in their LRR domains. The C-terminal region of GBP1 undergoes a large rotation compared to previously determined structures. We further show that the C-terminal farnesylation modification also plays a role in regulating GBP1 conformation. Our results suggest a general mechanism by which the IpaH proteins target their cellular substrates and shed light on the structural dynamics of the GBPs.

PH-Dependent Conformational Changes Due to Ionizable Residues in a Hydrophobic Protein Interior: The Study of L25K and L125K Variants of SNase.

Ionizable residues in the hydrophobic interior of certain proteins are known to play important roles in life processes like energy transduction and enzyme catalysis. These internal ionizable residues show experimental apparent pKa values having large shifts as compared to their values in solution. In the present work, we study the pH-dependent conformational changes undergone by two variants of staphylococcal nuclease (SNase), L25K and L125K, using pH replica exchange molecular dynamics (pH-REMD) in explicit solvent. Our results show that the observed pKa of Lys25 and Lys125 are significantly different than their pKa in solution. We observed that the internal lysine residues prefer to be water-exposed when protonated at low pH, but they remain buried within the hydrophobic pocket when deprotonated at high pH. Using thermodynamic laws, we estimate the microscopic conformation-specific pKa of the water-exposed and buried conformations of the internal lysine residues and explain their relation to the macroscopic observed pKa values. We present the differences in the microscopic mechanisms that lead to similar experimentally observed apparent pKa of Lys25 and Lys125, and explain the need of thermodynamic models of different complexities to account for our calculations. We see that L25K displays pH-dependent fluctuations throughout the entire β barrel and the α1 helix. In contrast, pH-independent fluctuations are observed in L125K, primarily limited to the α3 helix. The present computational study offers a detailed atomistic understanding of the determinants of the observed anomalous pKa of internal ionizable residues, bolstering the experimental findings.

Crystal structure of pentameric shell protein CsoS4B of Halothiobacillus neapolitanus α-carboxysome

Carboxysome, encapsulating an enzymatic core within an icosahedral-shaped semipermeable protein shell, could enhance CO2 fixation under low CO2 conditions in the environment. The shell of Halothiobacillus neapolitanus α-carboxysome possesses two 38% sequence-identical pentameric proteins, namely CsoS4A and CsoS4B. However, the functions of two paralogous pentameric proteins in α-carboxysome assembly remain unknown. Here we report the crystal structure of CsoS4B at 2.15 Å resolution. It displays as a stable pentamer, each subunit of which consists of a β-barrel core domain, in addition to an insertion of helix α1 that forms the central pore. Structural comparisons and multiple-sequence alignment strongly indicate that CsoS4A and CsoS4B differ from each other in interacting with various components of α-carboxysome, despite they share a similar overall structure. These findings provide the structural basis for further investigations on the self-assembly process of carboxysome.

Structural and functional insights of the capsid protein from Zika virus

Abstract Zika virus (ZIKV) emerged as a global public health concern, and is distinct from other flaviviruses in many aspects, e.g., causing transplacental infection, fetal abnormalities and vector-independent transmission through body fluids in humans. The capsid (C) protein is a multifunctional protein, since it binds to viral RNA in the process of nucleocapsid assembly and plays important roles in virus infection processes by interacting with cellular proteins, modulating cellular metabolism, apoptosis and immune response. Here we solved the crystal structure of ZIKV C protein at a resolution of 1.9 Å. The ZIKV C protein structure contains four α helices with a long pre-α1 loop, and forms dimers. The unique long pre-α1 loop in ZIKV C contributes to the tighter association of dimeric assembly and renders a divergent hydrophobic feature at the lipid bilayer interface in comparison with the known C structures of West Nile and dengue viruses. We reported the interaction between the ZIKV C protein and lipid droplets through confocal microscopy analysis. Substitutions of key amino acids in the pre-α1 loop of ZIKV C disrupted the interaction with lipid droplets, indicating the loop is critical for membrane association. We also recognized ZIKV C protein possesses broad binding capability to different nucleotide types, including single-stranded and double-stranded RNAs or DNAs. Furthermore, the highly positively charged interface, mainly formed by α4 helix, is proposed to be responsible for nucleotide binding. These findings will greatly enhance our understanding of ZIKV C protein, providing information for anti-ZIKV drug design targeting the C protein.

Polymorphism in chicken MHC-Y class I molecules that bind lipid ligands

Abstract Polymorphism is a major structural feature considered to set peptide-binding MHC class I molecules apart from other types of MHC class I molecules that bind other types of ligands. Most polymorphic residues in peptide-binding MHC class I molecules point into the ligand binding groove and delimit the types of peptides bound. For illustration, in an analysis of the α1 and α2 domains of 25 HLA class I sequences, most of the highly polymorphic positions (12 out of 15 positions with variability indices greater than six) have amino acids with side-chains pointing into or toward the ligand binding groove. In contrast, distribution of polymorphic positions in chicken MHC-Y class I molecules is strikingly different. An analysis of 25 MHC-Y class I molecules revealed 16 highly polymorphic positions (variability indices greater than six) in α1 and α2 domain sequences. Only seven of these have amino acids with side chains pointing into or toward the binding groove. The remaining polymorphic positions are located mostly along the α1 helix, pointing up and away from the binding groove. These findings are consistent with the MHC-Y class I binding groove being narrow and binding simple lipid ligands through hydrophobic interactions. It may be that the remaining surface polymorphism is the basis for specificity in the interactions of MHC-Y class I molecules in immune responses that remain to be defined. Support - USDA NIFA 2016-10247 & Caltech-City of Hope Biomedical Research Initiative

Crystal structure of the cold-adapted haloalkane dehalogenase DpcA from Psychrobacter cryohalolentis K5.

Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.

Lipid-modulation of membrane insertion and refolding of the apoptotic inhibitor Bcl-xL

Bcl-xL is a member of the Bcl-2 family of apoptotic regulators, responsible for inhibiting the permeabilization of the mitochondrial outer membrane, and a promising anti-cancer target. Bcl-xL exists in the following conformations, each believed to play a role in the inhibition of apoptosis: (a) a soluble folded conformation, (b) a membrane-anchored (by its C-terminal α8 helix) form, which retains the same fold as in solution and (c) refolded membrane-inserted conformations, for which no structural data are available. Previous studies established that in the cell Bcl-xL exists in a dynamic equilibrium between soluble and membranous states, however, no direct evidence exists in support of either anchored or inserted conformation of the membranous state in vivo. In this in vitro study, we employed a combination of fluorescence and EPR spectroscopy to characterize structural features of the bilayer-inserted conformation of Bcl-xL and the lipid modulation of its membrane insertion transition. Our results indicate that the core hydrophobic helix α6 inserts into the bilayer without adopting a transmembrane orientation. This insertion disrupts the packing of Bcl-xL and releases the regulatory N-terminal BH4 domain (α1) from the rest of the protein structure. Our data demonstrate that both insertion and refolding of Bcl-xL are modulated by lipid composition, which brings the apparent pKa of insertion to the threshold of physiological pH. We hypothesize that conformational rearrangements associated with the bilayer insertion of Bcl-xL result in its switching to a so-called non-canonical mode of apoptotic inhibition. Presented results suggest that the alteration in lipid composition before and during apoptosis can serve as an additional factor regulating the permeabilization of the mitochondrial outer membrane.

Molecular dynamics simulations study of influence of Tyr422Ala mutation on transcriptional enhancer activation domain 4 (TEAD4) and transcription co-activators complexes

Transcriptional enhancer activation domain (TEAD) proteins are the downstream transcriptional factor of the Hippo pathway. The transcription co-activators Yes-associated protein (YAP) and its paralog transcription co-activators with PDZ-binding motif (TAZ), binding to TEAD to promote transcription of genes in cell proliferation and anti-apoptosis, are key effectors of the Hippo pathway. TEAD4, one member of TEAD proteins, is specifically required in embryo implantation. The recently reported crystal structure of TEAD4-TAZ complex (PDB Code 5GN0) in mouse reveals that the interactions between the two helices of YAP/TAZ and TEAD4 are highly conserved. Point mutation of the residue Tyr422 of TEAD4 protein would disrupt the relevant hydrogen bond and even abolish the interaction. However, detailed information affected by the mutation at the atom level are still unrevealed. Molecular dynamics (MD) simulations and the molecular mechanics/Generalized-Born surface area (MM/GBSA) free energy calculations were used to explore the effects of mutation Tyr422Ala on the structural flexibility and conformational dynamics. The non-polar interactions play an indispensable role in the binding process of TEAD4 and YAP/TAZ. The helices α1 and α2 of YAP/TAZ provide a primary function to anchor YAP/TAZ well bound to TEAD4. The mutation Tyr422Ala disrupts the hydrogen-bonding network but do not obviously influence the secondary structure stability of TEAD4. The binding conformation of YAP/TAZ distorted by decreased non-polar interaction and the lost hydrogen bonds would lead to reduced interaction activity. The present study would provide important insights into the structure-function relationships of TEAD protein and give a new explanation for the affinity of YAP/TAZ with TEAD.

A Salt Bridge between α4 and α5 Helices Drives Differences in Flexibility and Potency of Inhibition among Regulator of G‐protein Signaling (RGS) Proteins

Regulators of G‐protein Signaling (RGS) proteins accelerate the rate of GTP hydrolysis, decreasing G‐protein signaling. Because there are many RGS isoforms with distinct tissue expressions, selective inhibition of RGS action may allow improved tissue specificity over global GPCR agonist application. RGS inhibitors discovered to date act by covalent modification of cysteine residues, and are specific for RGS4. However, single‐cysteine RGS mutants vary widely in their potency of inhibition by thiadiazolidinone (TDZD) inhibitors, with RGS19 being most potently inhibited. We have previously demonstrated a correlation between RGS protein flexibility and potency of inhibition by TDZDs. We hypothesized that differences in a salt bridge‐forming residue between the α4 and α5 helices drives differences in flexibility and potency of inhibition among RGS proteins.An acidic residue on the α4 helix that takes part in an α4‐α5 salt bride network is shared by RGS4 and RGS8 (D90 and E84 respectively), but not by RGS19 (L118). Upon mutation of RGS19 to induce formation of this salt bridge (L118D), the protein became 7°C more stable than WT, as measured by DSF. A mutation that removed this salt bridge in other isoforms caused an increase in melt temperature of 5°C in RGS4 (D90L) and a decrease in melt temperature of 8°C in RGS8 (E84L). Hydrogen‐deuterium exchange was used to evaluate the flexibility of mutant proteins. The α4 helix, which contains the mutation site, was found to be protected from deuterium exchange on addition of a salt bridge (RGS19 L118D), but have higher exchange compared to WT in both mutants where a salt bridge was removed (RGS4 D90L and RGS8 E84L). Changes in the α4 salt bridge‐forming residue also induced changes in the potency of inhibition of Gα binding by TDZD inhibitor CCG‐50014. RGS19 L118D had a lower potency of inhibition than WT RGS19, while mutations at RGS4 D90L and RGS8 E85L increased potency of inhibition compared to WT proteins. Together, these results show that differences in an α4 salt bridge‐forming residue are responsible for flexibility differences among RGS isoforms, and demonstrate a causal relationship between RGS protein flexibility and potency of TDZD inhibitors.Support or Funding InformationNSF grant 1507588This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.