NMR and Computational Characterization of Protein Structure and Ligand Binding

Nuclear magnetic resonance (NMR) techniques combined with computational methods such as docking and cheminformatics were used to characterize protein structure and ligand binding. The thioredoxin system of Mycobacterium tuberculosis consists of a thioredoxin reductase and at least three thioredoxins. This system is responsible for maintaining the cellular protein thiol redox state in normal state. This maintenance is important as the bacterium is engulfed by the human macrophage. Here it is bombarded by reactive oxygen and nitrogen species in an attempt to disrupt normal cellular function in part by perturbing the protein thiols. To this end, the solution structures of the three thioredoxins, A, B, and C, in the oxidized state were solved by NMR. Additionally, the reduced form of thioredoxin C was solved as well. Docking and NMR chemical shit pertubation experiments show promise for the inhibition of the thioredoxin C-thioredoxin reductase catalytic turnover. Automated docking is the process of computationally predicting how tightly a ligand binds to a protein and the correct orientation. The docking of an in-house collection of 10,590 chemicals into a protein called dual specificity phosphatase 5 identified potential ligands. These compounds were characterized as inhibitors in a phosphatase assay and as ligands in NMR chemical shift perturbation experiments. Based on a promising lead compound, additional chemicals were identified using cheminformatics and subjected to the same experimental verification

Development and validation of an improved algorithm for overlaying flexible molecules

A program for overlaying multiple flexible molecules has been developed. Candidate overlays are generated by a novel fingerprint algorithm, scored on three objective functions (union volume, hydrogen-bond match, and hydrophobic match), and ranked by constrained Pareto ranking. A diverse subset of the best ranked solutions is chosen using an overlay-dissimilarity metric. If necessary, the solutions can be optimised. A multi-objective genetic algorithm can be used to find additional overlays with a given mapping of chemical features but different ligand conformations. The fingerprint algorithm may also be used to produce constrained overlays, in which user-specified chemical groups are forced to be superimposed. The program has been tested on several sets of ligands, for each of which the true overlay is known from protein–ligand crystal structures. Both objective and subjective success criteria indicate that good results are obtained on the majority of these sets

Carbohydrate Recognition by an Architecturally Complex α-N-Acetylglucosaminidase from Clostridium perfringens

CpGH89 is a large multimodular enzyme produced by the human and animal pathogen Clostridium perfringens. The catalytic activity of this exo-α-d-N-acetylglucosaminidase is directed towards a rare carbohydrate motif, N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which is displayed on the class III mucins deep within the gastric mucosa. In addition to the family 89 glycoside hydrolase catalytic module this enzyme has six modules that share sequence similarity to the family 32 carbohydrate-binding modules (CBM32s), suggesting the enzyme has considerable capacity to adhere to carbohydrates. Here we suggest that two of the modules, CBM32-1 and CBM32-6, are not functional as carbohydrate-binding modules (CBMs) and demonstrate that three of the CBMs, CBM32-3, CBM32-4, and CBM32-5, are indeed capable of binding carbohydrates. CBM32-3 and CBM32-4 have a novel binding specificity for N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which thus complements the specificity of the catalytic module. The X-ray crystal structure of CBM32-4 in complex with this disaccharide reveals a mode of recognition that is based primarily on accommodation of the unique bent shape of this sugar. In contrast, as revealed by a series of X-ray crystal structures and quantitative binding studies, CBM32-5 displays the structural and functional features of galactose binding that is commonly associated with CBM family 32. The functional CBM32s that CpGH89 contains suggest the possibility for multivalent binding events and the partitioning of this enzyme to highly specific regions within the gastrointestinal tract

EVOLUTION OF THE KINETICS AND DYNAMICS OF HEME-CREVICE LOOP REGULATING CHEMISTRY IN HUMAN CYTOCHROME C

Cytochrome c, cytc, is a metalloprotein that plays primary roles in electron transport and intrinsic apoptotic pathways. Much of the chemistry that cytc is involved with is regulated by a highly conserved region known as the heme crevice loop, consisting of residues 70-85. Only three of these residues (those at positions 81, 83 and 85) are not universally conserved within the evolutionary timeline. Here I look to elucidate possible evolutionary roles for several of the key residues known to be important in regulating heme chemistry of cytc. I first address the role that lysine 72 plays in cytc folding and chemistry. Here I provide evidence that K72 alters the alkaline conformational transition of cytc. Trimethylated lysine 72, tmK72, was previously investigated and shows similar trends in peroxidase activity (McClelland et al 2013). Lastly I address I81 which is not only within the heme crevice loop and not universally conserved, but is also a hydrophobic surface residue. Here I make a Hu I81A variant, mutating to the alanine seen in yeast cytc. Our hypothesis was that this mutation would show a destabilization of the heme crevice loop region when monitoring the charge transfer band (695 nm), and more importantly observing an increase in peroxidase activity when monitoring for tetraguaiacol (470 nm) in our enzymatic assay. This signifies this mutation could have evolved to lock down that heme crevice loop in order to decrease peroxidase activity when intrinsic apoptosis pathways evolved in mammals. pH titration data showed a decrease in stability of the alkaline conformational transition in our I81A variant when compared to Hu WT. When looking at peroxidase activity we see a significant increase in kcat(s-1) values of I81A compared to Hu WT. The Hu I81A indeed shows what we would expect of a mutation which evolved to decrease peroxidase activity. Analysis of pH jump data in the Soret region of cytc shows that there is an effect on lysine 73 or 79 bound alkaline ligands (unable to be determined) by a decrease in amplitude, however there is no effect on the lysine 72 bound alkaline ligand

Computational Studies of Glycan Conformations in Glycoproteins

N-glycans refer to oligosaccharide chains covalently attached to the side chain of asparagine (Asn) residues, and the majority of proteins synthesized in the endoplasmic reticulum (ER) are N-glycosylated. N-glycans can modulate the structural properties of proteins due to their close proximity to their parent proteins and their interactions between the glycan and the protein surface residues. In addition, N-glycans provide specific regions of recognition for cellular and molecular recognition. Despite their biological importance, the structural understanding of glycans and the impact of glycosylation to glycan or protein structure are lacking. I have explored the conformational freedom of glycans and their conformational preferences in different environments using structural databases and computer simulations. First, I have developed an algorithm to reliably annotate a given atomic structure of glycans. This algorithm is important because many glycan molecules in the crystal structure database are misannotated or contain errors. Using the algorithm, a database of glycans found in the PDB is constructed and available to the public. Second, the impact of glycosylation on the glycan conformation has been examined. Contrary to the common belief that the glycan conformations are independent to the protein structure, it appears that the protein structure can significantly affect the glycan structure upon glycosylation. This observation is significant because it may provide insight into protein-glycan interaction and opens up the possibility of a template-based glycan modeling approach. Third, the differences in conformational preference between glycans in solution and in glycoproteins has been examined. Using molecular dynamics (MD) simulations, the conformational preference of N-glycan pentassacharide in solution is exhaustively studied. Surprisingly, the conformational distribution is dominated by a single major conformational state and several minor conformational states. The dominant conformational state adopts a more extended conformation, thus it appears that entropy plays an important role in determining the conformational state. On the other hand, in glycoproteins, glycans can interact with surrounding protein side chains and, as a result, several conformational states are more equally populated. Based on these observations, a protocol is proposed for modeling the glycan portion of a known protein structure. It is typically more managable to acquire an atomic resolution structure or aglycoprotein (glycoprotein without glycan). In addition, the glycoform and the glycosylation site can be identified independently by mass spectrometry or NMR. The proposed modeling protocol assumes the glycosylation site, glycoform, and aglycoprotein structure are already known, and builds glycan structure models on top of the known aglycoprotein structure. The performance of the modeling protocol is greatly improved by using appropriate template structures. This protocol can be used to generate the initial model for MD simulations or refinement of low resolution models from experiments (small angle X-ray scattering and electron microscopy)

Dissecting the Roles of Dynamic Substructures in Beta-Barrel Containing Coactivators

Starting off the central dogma of genetics, transcription is an integral process in all of life. Transcription is highly regulated, with many players and moving parts, such as transcription factors that localize transcriptional machinery to the promoter, and regulators that modulate expression levels. Despite being well studied, there lacks a mechanistic understanding of how binding occur, largely due to the difficult nature of studying these interactions. Intrinsic disorder, transient interactions, and highly dynamic, protein-protein interactions at the site of transcription are difficult systems to study mechanistically and structurally. Due to their critical role in all of life, it is crucial to understand the molecular recognition details of these interactions. Molecular recognition models of activator-coactivator interactions were argued to be largely nonspecific, dictated by unstructured and negatively charged transcriptional activators bound to DNA interacting with amphipathic coactivators. However, this model does not accurately represent the critical role of activator-coactivator interactions. Put another way, it is too simplified. The work in this thesis aims to decipher the molecular recognition mechanisms of coactivators recognizing activator binding partners using a beta-barrel containing coactivator activator binding domain termed Activator Interacting Domain (AcID). Recent data has shifted the paradigm of molecular recognition to one that is more specific. Originally demonstrated in KIX, it has been observed that conformational changes are induced upon activator binding to the activator binding domain AcID of the Mediator complex subunit, Med25. Moreover, it was shown that despite overlapping binding sites, unique conformational ensembles were observed for each binding partner. Using a second protein containing two tandem AcID motifs, the work in this thesis aimed to expand upon this conservation of molecular recognition. We show that the AcIDs are capable of recognizing overlapping binding partners in vitro, demonstrating that selectivity is achieved through means other than activator sequence. Using transient kinetics, binding mechanisms of the different AcID motifs were observed. Specifically, despite being paralogs, it was found that different binding modes were observed, suggesting that changes in overall dynamic resulted in conformations specific to each AcID motif without drastically changing the overall binding affinities. Further, it has been demonstrated that an allosteric network exists between the binding faces of activator binding domains within coactivtors. Using a kinetic approach, we demonstrated that there is allosteric communication within the different AcID motifs. Similar to how conformational changes of activator binding domains are mediated through dynamic substructures, allosteric communication is also mediated by loops and helices. These dynamic substructures can be exploited as hotspots for targeting, as these allosteric regions are not as highly conserved in paralogs. We demonstrate that identified allosteric modulators can be used as chemical probes to perturb the dynamic hotspots, providing an opportunity to target homologous proteins with high selectivity. Further, we show that even highly related activators are able to induce differential conformations in activator binding domains, highlighting that these interactions are specific. Using a biophysical and biochemical approach, the work in this dissertation demonstrates that activator binding domains are capable of sharing a conserved binding mechanism. By demonstrating that differences in dynamic substructures flanking the binding faces can induce differential conformational changes, we provide a mechanism by which molecular recognition can occur. We highlight that conformational plasticity can influence allosteric communication and provide an opportunity to selectively target activator-coactivator interactions.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/169813/1/sdesalle_1.pd

Structure based drug design for the discovery of promising inhibitors of human Bcl-2 and Streptococcus dysgalactiae LytR proteins

Drug research has evolved significantly in the last decades toward the concept of the rational design of drugs. The capability to study molecular interactions at the atomic level and to rationalize this knowledge to construct and improve drug candidates provided the premises of structure-based drug design (SBDD). This approach allied to the computational methods available nowadays yields the opportunity to expedite the intricate process of drug discovery. In the present thesis, the SBDD approach was implemented to study promising candidate inhibitors of the human Bcl-2 and the Streptococcus dysgalactiae LytR proteins. Half of the cancers in humans are estimated to be related with overexpression of Bcl-2 protein. This macromolecule is responsible for the inhibition of the apoptotic process, which is pivotal for the elimination of abnormal cells. When Bcl-2 is overexpressed, these abnormal cells don’t respond to death stimuli, either endogenous or exogenous, such as chemotherapeutic, and become immortal. Promising 4H-chromene and indole derivatives were studied regarding their potential to inhibit Bcl-2. Molecular docking studies revealed sub-micromolar binding of the 4Hchromene activemethine and the indole derivatives in the binding groove essential for Bcl-2 biological function. Biophysical characterization did not demonstrate significant evidence of binding between Bcl-2 and the compounds under study, probably due to their small network of interactions with the binding pocket residues. The structure determination process of the proteinligand complexes achieved preliminary co-crystallization conditions that require further optimization. Numerous infectious diseases are associated to the bacterial biofilm phenotype, which consists of agglomerates of cells enclosed in a self-produced matrix. Biofilms confer bacteria improved resistance to the host’s innate immune system and to conventional antibiotics. LytR belongs to the LCP family of proteins, which are thought to be responsible for the attachment of anionic polymers to the peptidoglycan, protecting the Gram-positive bacteria from phagocytosis and lysis. Previous virtual screening studies yielded ellagic acid and fisetin has promising inhibitors of LytR, displaying anti-biofilm activity. Molecular docking revealed binding of these compounds in the hypothetical active site of LytR, with micromolar affinities, and specific interactions with crucial protein residues for catalysis. Biophysical techniques failed to provide evidence of protein-ligand interactions, although this may be related to the possible co-purification with a lipidic substrate, which has been reported before. Mass spectrometry or structural determination, through X-ray crystallography or NMR, should be pivotal to establish evidence of this molecule’s accommodation in the binding pocket

A poised fragment library enables rapid synthetic expansion yielding the first reported inhibitors of PHIP(2), an atypical bromodomain

Research into the chemical biology of bromodomains has been driven by the development of acetyl-lysine mimetics. The ligands are typically anchored by binding to a highly conserved asparagine residue. Atypical bromodomains, for which the asparagine is mutated, have thus far proven elusive targets, including PHIP(2) whose parent protein, PHIP, has been linked to disease progression in diabetes and cancers. The PHIP(2) binding site contains a threonine in place of asparagine, and solution screening have yielded no convincing hits. We have overcome this hurdle by combining the sensitivity of X-ray crystallography, used as the primary fragment screen, with a strategy for rapid follow-up synthesis using a chemically-poised fragment library, which allows hits to be readily modified by parallel chemistry both peripherally and in the core. Our approach yielded the first reported hit compounds of PHIP(2) with measurable IC50 values by an AlphaScreen competition assay. The follow-up libraries of four poised fragment hits improved potency into the sub-mM range while showing good ligand efficiency and detailed structural data

Alternative protein conformations: yeast iso-1-cytochrome c and heme crevice dynamics

The field of protein biochemistry has been dominated by the dogma that a protein sequence yields a 3-dimensional structure important for a singular function. More modern insights are beginning to demonstrate that proteins are not static structures. Rather, proteins undergo numerous conformational fluctuations yielding an ensemble of conformational populations. Conformational change can result in changed or altered protein function. Small or large energetic barriers existing between conformers regulate the ease with which a protein can sample alternative conformations. In the dissertation work presented here, alternative conformations of yeast iso-1-cytochrome c are investigated with particular emphasis on heme crevice loop dynamics. The heme crevice loop, or O-loop D, is a highly conserved, dynamic region. Conformational changes in O-loop D lead to altered electron transfer and peroxidase activity in cytochrome c (Cytc). As Cytc participates in both the electron transport chain and functions as a peroxidase during apoptosis, it is important to understand how this conformational change is regulated. Within O-loop D we investigate the effects of a trimethyllysine to alanine mutation and a destabilizing leucine to alanine mutation at residues 72 and 85, respectively, on heme crevice dynamics. Residue 72 plays an important role in regulating access to alternative heme crevice conformers. Of particular interest, residue 72 plays a role in regulating access to a peroxidase capable conformer of Cytc, a function of Cytc during the early stages of apoptosis. We have also solved the structure of the first monomeric Cytc structure in a peroxidase capable conformer, as well as, a dimeric Cytc structure with CYMAL-6 protruding into the interior of the heme cavity, in a manner potentially similar to the Cytc/cardiolipin interaction