Structure Guided Design of Bacteriophage Qß Mutants as Next Generation Carriers for Conjugate Vaccines.

Vaccines are critical tools to treat and prevent diseases. For an effective conjugate vaccine, the carrier is crucial, but few carriers are available for clinical applications. In addition, a drawback of current protein carriers is that high levels of antibodies against the carrier are induced by the conjugate vaccine, which are known to interfere with the immune responses against the target antigen. To overcome these challenges, we obtained the near atomic resolution crystal structure of an emerging protein carrier, i.e., the bacteriophage Qß virus like particle. On the basis of the detailed structural information, novel mutants of bacteriophage Qß (mQß) have been designed, which upon conjugation with tumor associated carbohydrate antigens (TACAs), a class of important tumor antigens, elicited powerful anti-TACA IgG responses and yet produced lower levels of anticarrier antibodies as compared to those from the wild type Qß-TACA conjugates. In a therapeutic model against an aggressive breast cancer in mice, 100% unimmunized mice succumbed to tumors in just 12 days even with chemotherapy. In contrast, 80% of mice immunized with the mQß-TACA conjugate were completely free from tumors. Besides TACAs, to aid in the development of vaccines to protect against COVID-19, the mQß based conjugate vaccine has been shown to induce high levels of IgG antibodies against peptide antigens from the SARS-CoV-2 virus, demonstrating its generality. Thus, mQß is a promising next-generation carrier platform for conjugate vaccines, and structure-based rational design is a powerful strategy to develop new vaccine carriers.

An Interhelical Salt Bridge Controls Flexibility and Inhibitor Potency for Regulators of G-protein Signaling Proteins 4, 8, and 19.

Regulators of G-protein signaling (RGS) proteins modulate receptor signaling by binding to activated G-protein α-subunits, accelerating GTP hydrolysis. Selective inhibition of RGS proteins increases G-protein activity and may provide unique tissue specificity. Thiadiazolidinones (TDZDs) are covalent inhibitors that act on cysteine residues to inhibit RGS4, RGS8, and RGS19. There is a correlation between protein flexibility and potency of inhibition by the TDZD 4-[(4- fluorophenyl)methyl]-2-(4-methylphenyl)-1,2,4-thiadiazolidine-3,5-dione (CCG-50014). In the context of a single conserved cysteine residue on the α 4 helix, RGS19 is the most flexible and most potently inhibited by CCG-50014, followed by RGS4 and RGS8. In this work, we identify residues responsible for differences in both flexibility and potency of inhibition among RGS isoforms. RGS19 lacks a charged residue on the α 4 helix that is present in RGS4 and RGS8. Introducing a negative charge at this position (L118D) increased the thermal stability of RGS19 and decreased the potency of inhibition of CCG-50014 by 8-fold. Mutations eliminating salt bridge formation in RGS8 and RGS4 decreased thermal stability in RGS8 and increased potency of inhibition of both RGS4 and RGS8 by 4- and 2-fold, respectively. Molecular dynamics simulations with an added salt bridge in RGS19 (L118D) showed reduced RGS19 flexibility. Hydrogen-deuterium exchange studies showed striking differences in flexibility in the α 4 helix of RGS4, 8, and 19 with salt bridge-modifying mutations. These results show that the α 4 salt bridge-forming residue controls flexibility in several RGS isoforms and supports a causal relationship between RGS flexibility and the potency of TDZD inhibitors. SIGNIFICANCE STATEMENT: Inhibitor potency is often viewed in relation to the static structure of a target protein binding pocket. Using both experimental and computation studies we assess determinants of dynamics and inhibitor potency for three different RGS proteins. A single salt bridge-forming residue determines differences in flexibility between RGS isoforms; mutations either increase or decrease protein motion with correlated alterations in inhibitor potency. This strongly suggests a causal relationship between RGS protein flexibility and covalent inhibitor potency.

Interplay of cysteine exposure and global protein dynamics in small-molecule recognition by a regulator of G-protein signaling protein.

Regulator of G protein signaling (RGS) proteins play a pivotal role in regulation of G protein-coupled receptor (GPCR) signaling and are therefore becoming an increasingly important therapeutic target. Recently discovered thiadiazolidinone (TDZD) compounds that target cysteine residues have shown different levels of specificities and potencies for the RGS4 protein, thereby suggesting intrinsic differences in dynamics of this protein upon binding of these compounds. In this work, we investigated using atomistic molecular dynamics (MD) simulations the effect of binding of several small-molecule inhibitors on perturbations and dynamical motions in RGS4. Specifically, we studied two conformational models of RGS4 in which a buried cysteine residue is solvent-exposed due to side-chain motions or due to flexibility in neighboring helices. We found that TDZD compounds with aromatic functional groups perturb the RGS4 structure more than compounds with aliphatic functional groups. Moreover, small-molecules with aromatic functional groups but lacking sulfur atoms only transiently reside within the protein and spontaneously dissociate to the solvent. We further measured inhibitory effects of TDZD compounds using a protein-protein interaction assay on a single-cysteine RGS4 protein showing trends in potencies of compounds consistent with our simulation studies. Thermodynamic analyses of RGS4 conformations in the apo-state and on binding to TDZD compounds revealed links between both conformational models of RGS4. The exposure of cysteine side-chains appears to facilitate initial binding of TDZD compounds followed by migration of the compound into a bundle of four helices, thereby causing allosteric perturbations in the RGS/Gα protein-protein interface.

Differential Protein Dynamics of Regulators of G-Protein Signaling: Role in Specificity of Small-Molecule Inhibitors.

Small-molecule inhibitor selectivity may be influenced by variation in dynamics among members of a protein family. Regulator of G-protein Signaling (RGS) proteins are a family that plays a key role in G-Protein Coupled Receptor (GPCR) signaling by binding to active Gα subunits and accelerating GTP hydrolysis, thereby terminating activity. Thiadiazolidinones (TDZDs) inhibit the RGS-Gα interaction by covalent modification of cysteine residues in RGS proteins. Some differences in specificity may be explained by differences in the complement of cysteines among RGS proteins. However, key cysteines shared by RGS proteins inhibited by TDZDs are not exposed on the protein surface, and differences in potency exist among RGS proteins containing only buried cysteines. We hypothesize that differential exposure of buried cysteine residues among RGS proteins partially drives TDZD selectivity. Hydrogen-deuterium exchange (HDX) studies and molecular dynamics (MD) simulations were used to probe the dynamics of RGS4, RGS8, and RGS19, three RGS proteins inhibited at a range of potencies by TDZDs. When these proteins were mutated to contain a single, shared cysteine, RGS19 was found to be most potently inhibited. HDX studies revealed differences in α4 and α6 helix flexibility among RGS isoforms, with particularly high flexibility in RGS19. This could cause differences in cysteine exposure and lead to differences in potency of TDZD inhibition. MD simulations of RGS proteins revealed motions that correspond to solvent exposure observed in HDX, providing further evidence for a role of protein dynamics in TDZD selectivity.

Movement disorder in GNAO1 encephalopathy associated with gain-of-function mutations.

OBJECTIVE: To define molecular mechanisms underlying the clinical spectrum of epilepsy and movement disorder in individuals with de novo mutations in the GNAO1 gene. METHODS: We identified all GNAO1 mutations reported in individuals with epilepsy (early infantile epileptiform encephalopathy 17) or movement disorders through April 2016; 15 de novo mutant alleles from 25 individuals were introduced into the Gαo subunit by site-directed mutagenesis in a mammalian expression plasmid. We assessed protein expression and function in vitro in HEK-293T cells by Western blot and determined functional Gαo-dependent cyclic adenosine monophosphate (cAMP) inhibition with a coexpressed α2A adrenergic receptor. RESULTS: Of the 15 clinical GNAO1 mutations studied, 9 show reduced expression and loss of function (LOF; <90% maximal inhibition). Six other mutations show variable levels of expression but exhibit normal or even gain-of-function (GOF) behavior, as demonstrated by significantly lower EC50 values for α2A adrenergic receptor-mediated inhibition of cAMP. The GNAO1 LOF mutations are associated with epileptic encephalopathy while GOF mutants (such as G42R, G203R, and E246K) or normally functioning mutants (R209) were found in patients with movement disorders with or without seizures. CONCLUSIONS: Both LOF and GOF mutations in Gαo (encoded by GNAO1) are associated with neurologic pathophysiology. There appears to be a strong predictive correlation between the in vitro biochemical phenotype and the clinical pattern of epilepsy vs movement disorder.