Unraveling infectious structures, strain variants and species barriers for the yeast prion [PSI+]

Prions are proteins that can access multiple conformations, at least one of which is beta-sheet rich, infectious and self-perpetuating in nature. These infectious proteins show several remarkable biological activities, including the ability to form multiple infectious prion conformations, also known as strains or variants, encoding unique biological phenotypes, and to establish and overcome prion species (transmission) barriers. In this Perspective, we highlight recent studies of the yeast prion [PSI+], using various biochemical and structural methods, that have begun to illuminate the molecular mechanisms by which self-perpetuating prions encipher such biological activities. We also discuss several aspects of prion conformational change and structure that remain either unknown or controversial, and we propose approaches to accelerate the understanding of these enigmatic, infectious conformers

Leveraging Structural Flexibility to Predict Protein Function

Proteins are essentially versatile and flexible molecules and understanding protein function plays a fundamental role in understanding biological systems. Protein structure comparisons are widely used for revealing protein function. However,with rigidity or partial rigidity assumption, most existing comparison methods do not consider conformational flexibility in protein structures. To address this issue, this thesis seeks to develop algorithms for flexible structure comparisons to predict one specific aspect of protein function, binding specificity. Given conformational samples as flexibility representation, we focus on two predictive problems related to specificity: aggregate prediction and individual prediction.For aggregate prediction, we have designed FAVA (Flexible Aggregate Volumetric Analysis). FAVA is the first conformationally general method to compare proteins with identical folds but different specificities. FAVA is able to correctly categorize members of protein superfamilies and to identify influential amino acids that cause different specificities. A second method PEAP (Point-based Ensemble for Aggregate Prediction) employs ensemble clustering techniques from many base clustering to predict binding specificity. This method incorporates structural motions of functional substructures and is capable of mitigating prediction errors.For individual prediction, the first method is an atomic point representation for representing flexibilities in the binding cavity. This representation is able to predict binding specificity on each protein conformation with high accuracy, and it is the first to analyze maps of binding cavity conformations that describe proteins with different specificities. Our second method introduces a volumetric lattice representation. This representation localizes solvent-accessible shape of the binding cavity by computing cavity volume in each user-defined space. It proves to be more informative than point-based representations. Last but not least, we discuss a structure-independent representation. This representation builds a lattice model on protein electrostatic isopotentials. This is the first known method to predict binding specificity explicitly from the perspective of electrostatic fields.The methods presented in this thesis incorporate the variety of protein conformations into the analysis of protein ligand binding, and provide more views on flexible structure comparisons and structure-based function annotation of molecular design

Unraveling the Allosteric Mechanism of Serine Protease Inhibition by an Antibody

SummaryRecent structural studies have outlined the mechanism of protease inhibition by active site-directed antibodies. However, the molecular basis of allosteric inhibition by antibodies has been elusive. Here we report the 2.35 Å resolution structure of the trypsin-like serine protease hepatocyte growth factor activator (HGFA) in complex with the allosteric antibody Ab40, a potent inhibitor of HGFA catalytic activity. The antibody binds at the periphery of the substrate binding cleft and imposes a conformational change on the entire 99-loop (chymotrypsinogen numbering). The altered conformation of the 99-loop is incompatible with substrate binding due to the partial collapse of subsite S2 and the reorganization of subsite S4. Remarkably, a single residue deletion of Ab40 abolished inhibition of HGFA activity, commensurate with the reversal of the 99-loop conformation to its “competent” state. The results define an “allosteric switch” mechanism as the basis of protease inhibition by an allosteric antibody

Structural interrogation of phosphoproteome identified by mass spectrometry reveals allowed and disallowed regions of phosphoconformation

High-throughput mass spectrometric (HT-MS) study is the method of choice for monitoring global changes in proteome. Data derived from these studies are meant for further validation and experimentation to discover novel biological insights. Here we evaluate use of relative solvent accessible surface area (rSASA) and DEPTH as indices to assess experimentally determined phosphorylation events deposited in PhosphoSitePlus. Based on accessibility, we map these identifications on allowed (accessible) or disallowed (inaccessible) regions of phosphoconformation. Surprisingly a striking number of HT- MS/MS derived events (1461/5947 sites or 24.6%) are present in the disallowed region of conformation. By considering protein dynamics, autophosphorylation events and/or the sequence specificity of kinases, 13.8% of these phosphosites can be moved to the allowed region of conformation. We also demonstrate that rSASA values can be used to increase the confidence of identification of phosphorylation sites within an ambiguous MS dataset. While MS is a stand-alone technique for the identification of vast majority of phosphorylation events, identifications within disallowed region of conformation will benefit from techniques that independently probe for phosphorylation and protein dynamics. Our studies also imply that trapping alternate protein conformations may be a viable alternative to the design of inhibitors against mutation prone drug resistance kinases

Specificity Determination by paralogous winged helix-turn-helix transcription factors

Transcription factors (TFs) localize to regulatory regions throughout the genome, where they exert physical or enzymatic control over the transcriptional machinery and regulate expression of target genes. Despite the substantial diversity of TFs found across all kingdoms of life, most belong to a relatively small number of structural families characterized by homologous DNA-binding domains (DBDs). In homologous DBDs, highly-conserved DNA-contacting residues define a characteristic ‘recognition potential’, or the limited sequence space containing high-affinity binding sites. Specificity-determining residues (SDRs) alter DNA binding preferences to further delineate this sequence space between homologous TFs, enabling functional divergence through the recognition of distinct genomic binding sites. This thesis explores the divergent DNA-binding preferences among dimeric, winged helix-turn-helix (wHTH) TFs belonging to the OmpR sub-family. As the terminal effectors of orthogonal two-component signaling pathways in Escherichia coli, OmpR paralogs bind distinct genomic sequences and regulate the expression of largely non-overlapping gene networks. Using high-throughput SELEX, I discover multiple sources of variation in DNA-binding, including the spacing and orientation of monomer sites as well as a novel binding ‘mode’ with unique half-site preferences (but retaining dimeric architecture). Surprisingly, given the diversity of residues observed occupying positions in contact with DNA, there are only minor quantitative differences in sequence-specificity between OmpR paralogs. Combining phylogenetic, structural, and biological information, I then define a comprehensive set of putative SDRs, which, although distributed broadly across the protein:DNA interface, preferentially localize to the major groove of the DNA helix. Direct specificity profiling of SDR variants reveals that individual SDRs impact local base preferences as well as global structural properties of the protein:DNA complex. This study demonstrates clearly that OmpR family TFs possess multiple ‘axes of divergence’, including base recognition, dimeric architecture, and structural attributes of the protein:DNA complex. It also provides evidence for a common structural ‘code’ for DNA-binding by OmpR homologues, and demonstrates that surprisingly modest residue changes can enable recognition of highly divergent sequence motifs. Importantly, well-characterized genomic binding sites for many of the TFs in this study diverge substantially from the presented de novo models, and it is unclear how mutations may affect binding in more complex environments. Further analysis using native sequences is required to build combined models of cis- and trans-evolution of two-component regulatory networks

Coarse-grained models for Protein Folding and Function

The plan of this thesis is as follows: in the next chapter (chapter 1) we review the main properties of globular proteins, in particular focusing on the state of the art of protein folding and design. Then we describe in detail the simple model for folding adopted throughout the present work (chapter 2). Chapter 3 shows the further modeling introduced to handle the specific subject of disordered proteins, with full explanation of all parameters used and with some possible interpretation of the results obtained. In the last chapter of this work (chapter 4) we present a study on the near equilibrium dynamics of two small proteins in the family of truncated hemoglobins, developed under the framework of a Gaussian network approach

Intramolecular hydrogen-bonding in aqueous carbohydrates as a cause or consequence of conformational preferences: a molecular dynamics study of cellobiose stereoisomers

It is often assumed that intramolecular hydrogen-bonding (H-bonding) exerts a significant influence on the conformational properties of aqueous (bio-)polymers. To discuss this statement, one should, however, distinguish between solvent-exposed and buried H-bonds, and between their respective roles in promoting stability (i.e., as a driving force) and specificity (for which the term steering force is introduced here). In this study, the role of solvent-exposed H-bonding in carbohydrates as a driving or steering force is probed using explicit-solvent molecular dynamics simulations with local elevation umbrella sampling in the simple context of cellobiose stereoisomers. More specifically, four β(1→4)-linked d-aldohexopyranose disaccharides are considered, which present a different stereochemisty of the potentially H-bonding groups neighboring the glycosidic linkage. Although the epimerization may largely alter the intramolecular trans-glycosidic H-bonding pattern, it is found to have only very limited influence on the Ramachandran free-energy map of the disaccharide, a loss of intramolecular H-bonding being merely compensated for by an enhancement of the interaction with the solvent molecules. This finding suggests that solvent-exposed trans-glycosidic H-bonding (and in particular the $$\hbox{HO}_3^{\prime}$$ →O5 H-bond) is not the cause of the 21-helical secondary structure characteristic of cellooligosaccharides, but rather the opportunistic consequence of a sterically and stereoelectronically dictated conformational preference. In other words, for these compounds, solvent-exposed H-bonding appears to represent a minor (possibly adverse) conformational driving as well as steering forc