No description available.

abstract: Integrins are a family of αβ heterodimeric transmembrane receptors. As an important class of adhesion receptors, integrins mediate cell adhesion, migration, and transformation through bidirectional signaling across the plasma membrane. Among the 24 different types of integrins, which are notorious for their capacity to recognize multiple ligands, the leukocyte integrin αMβ2 (Mac-1) is the most promiscuous member. In contrast to other integrins, Mac1 is unique with respect to its preference for cationic ligands. In this thesis, a new Mac-1 cationic ligand named pleiotrophin (PTN) is uncovered. PTN is an important cytokine and growth factor. Its activities in mitogenesis and angiogenesis have been extensively researched, but its function on immune cells was not widely explored. In this research, the cell biology and biochemical evidences show that PTN can regulate various Mac-1-expressing cells functions through the activation of the extracellular signal regulated kinases. Direct interactions between PTN and the αM I-domain, the major ligand-binding domain of Mac-1, has been shown using biolayer interferometry analyses and confirmed by solution NMR spectroscopy. The binding epitopes and the binding mechanism of PTN and αM I-domain interaction were further revealed by peptide array analysis and microscale thermophoresis. The data suggested that PTN’s thrombospondin type-1 repeat (TSR) domains and αM I-domain metal-ion-dependent adhesion site (MIDAS) are the major binding sites. In addition, this interaction followed a novel metal-ion independent binding mechanism which has not been found in other integrins. After a series of characterizations of αM I-domain using both experimental and computational methods, it showed that activated αM I-domain is significantly more dynamic than inactive αM I-domain, and the dynamics seem to modulate the effect of Mg2+ on its interactions with cationic ligands. To further explore the PTN induced Mac-1 structure rearrangement, intact Mac-1 was studied by negative stain electron microscopy. The results showed that the Mac-1 exhibited a very heterogeneous conformation distribution in detergents. In contrast, the Mac-1 adopted predominantly the bent conformation in phospholipid nanodisc condition. This Mac-1 nanodisc model provides a new platform for studying intact Mac-1 activation mechanism in a more physiologically relevant manner in the future. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

Pas de résumé / No abstract

No description available.

Citrus and other fruits produce secondary metabolites that are synthesized, regulated, and modified by a class of enzymes called glucosyltransferases. This class of enzymes is of interest to this lab due to their unique structural and functional properties. Glucosides of flavonoids produced by glucosyltransferases are a critical part of plant metabolism and survival; many have health benefits when consumed. One such glucosyltransferase, found in Duncan grapefruit (Citrus paradisi), was identified, recombinantly expressed, and shown through biochemical characterization to exclusively glucosylate the flavonol class of flavonoids at the 3-OH position. The structural basis that accounts for a glucosyltransferase’s selectivity is not currently known, however great advances have been realized through the protein crystallization of 6 different secondary product glucosyltransferases. None of these show the same specificity exhibited by this flavonol-specific glucosyltransferase, CP3GT. The WT enzyme and two mutants have undergone site-directed mutagenesis to insert thrombin cleavage sites for removal of recombinant protein tags. The plasmid was transformed into yeast and protein was expressed through methanol induction. Cobalt column affinity chromatography was used to purify the protein. An aliquot of protein was treated with thrombin to remove tags and both tagged and native protein were assayed for activity with the flavonol quercetin. The data show that the reaction is linear for at least 15 minutes when 2ug of enzyme is used. Thus, kinetics assays will be conducted for 10 minutes. The presence of tags on the enzyme does not appear to impact activity with respect to the time course, however, more assays must be conducted to reliable confirm this with kinetic assays under different conditions. It is hypothesized that obtaining a crystal structure for this enzyme will illuminate the structural basis of its specificity. Additionally, it is hypothesized that a thrombin cleavage gene vector inserted for removal of purification tags will have no impact on enzyme activity or specificity.

GLUT1-mediated, facilitated sugar transport is proposed to be an example of transport by a carrier that alternately presents exofacial (e2) and endofacial (e1) substrate binding sites, commonly referred to as the alternating access carrier model. This hypothesis is incompatible with observations of co-existent exo- and endofacial ligand binding sites, transport allostery, and e1 ligand (e.g. cytochalasin B) induced GLUT1 sugar occlusion. The fixed-site carrier model proposes co-existent, interacting e2 and e1 ligand binding sites but involves sugar translocation by geminate exchange through internal cavities. Demonstrations of membrane-resident dimeric and tetrameric GLUT1 and of e2, e1 and occluded GLUT conformations in GLUT crystals of monodisperse, detergent-solubilized proteins suggest a third model. Here, GLUT1 is an alternating access carrier but the transporter complex is a dimer of GLUT1 dimers, in which subunit interactions produce two e2 and two e1 conformers at any instant. The crystallographic structures in different conformations can be utilized to further understand the transport cycle, ligand binding behavior and complex kinetics observed in GLUT1. Specifically, the GLUT1 crystal structure and homology models based upon related major facilitator superfamily proteins were used in this study, to understand inhibitor binding, ligand binding induced GLUT1 transport allostery and the existence of helix packing/oligomerization motifs and glycine induced flexibility. These studies suggest that GLUT1 functions as an oligomeric allosteric carrier where cis-allostery is an intramolecular behavior and trans-allostery is an intermolecular behavior. Additionally, mutations of a dynamic glycine affect the turnover of the transporter while mutations to helix packing motifs affect affinity.

Rab11 is a critical GTPase involved in the regulation of membrane trafficking in the endocytic pathway, and it’s misregulation is involved in a variety of human diseases including Huntington’s disease and Alzheimer’s disease. Additionally, de novo mutations (DNMs) of Rab11 have been identified in patients with developmental disorders, and interestingly several parasites, viruses, and bacteria can subvert membrane trafficking through Rab11 positive vesicles to allow for replication and evasion from the immune system. Although Rab11 is one of the best characterized Rab GTPases, hindering the capability to completely understand Rab11 regulation and its role in human disease is the lack of detail describing how Rab11 proteins are activated by their cognate guanine nucleotide exchange factors (GEFs). This thesis is therefore focused on revealing the molecular mechanisms of the GEFs responsible for the activation of Rab11: SH3BP5 and TRAPPII. To investigate the recently discovered GEF SH3BP5, we solved the 3.1Å structure of Rab11 bound to SH3BP5 and revealed a coiled coil architecture of SH3BP5 that mediates exchange through a unique Rab-GEF interaction. The structure revealed a unique rearrangement of the switch-I region of Rab11 compared to other solved Rab-GEF structures, with a constrained conformation when bound to SH3BP5. Mutational analysis of switch-I revealed the molecular determinants that allow for Rab11 selectivity over evolutionarily similar Rab GTPases, and GEF deficient mutants of SH3BP5 show greatly decreased Rab11 activation in cellular assays of active Rab11. To interrogate the highly controversial GEF TRAPPII, we recombinantly expressed and purified the 9 subunit, 427 kDa complex in Spodoptera frugiperda 9(Sf9) cells. We found that the TRAPPII complex is a GEF for both Rab1 and Rab11, and we discovered novel activity for another Rab GTPase. To interrogate the role of these GEFs in human disease, we used HDX-MS and nucleotide exchange assays to show that some DNMs destabilize Rab11 either through a complete or partial disruption of nucleotide binding. Importantly, we discovered that one of these DNMs, K13N, completely prevented SH3BP5 and TRAPPII mediated nucleotide exchange, revealing a putative mechanism of disease. Overall the work completed in this thesis leads to a greater understanding of the molecular mechanisms underlying the activation of Rab11 by its cognate GEFs. / Graduate / 2020-11-25

<p>  </p> <p>Proteins are the active biomolecules of the cell. They perform metabolic action, give the cell structure, protect the cell from antigens, give the cell motility, and much more. The function of proteins are intrinsically linked to their structures, so it is therefore necessary to characterize the structure of a protein to fully understand its function and operation. In this research the application of computational methods, primarily molecular dynamics, towards protein structure determination, refinement, and quality assessment were studied. I applied molecular dynamics techniques to four major projects; the determination of relative error of atomic models deposited with electron microscopy maps in the EMDB, solving and refining atomics structure models for the PhageG major capsid proteins, the elucidation of the structure the protein USP7 and the binding pose of a of a candidate therapeutic drug, and the determination of relative stability of candidate protein folds to distinguish near native models from not. Each year an increasing number of protein structures have been solved using electron microscopy (EM). The influx of solved structure has proven to be a boon to the community, but it is necessary to note that the quality EM maps vary substantially. To understand to what extent atomic structure models generated from EM matched their respective maps, two computational structure refinement methods were used to examine how much structures could be refined. The deviation from the starting structure by refinement, as well as the disagreement between refined models produced by the two computational methods, scaled inversely with both the global and local map resolutions. The results suggested that the observed discrepancy between the deposited maps and refined models is due to the lack of resolvable structural data present in EM maps at low to moderate resolutions, and therefore these annotations must be used with caution in further applications. I also successfully implemented molecular dynamics as a method for protein structure quality assessment. Proteins tend towards shapes which minimize their energy. Experimentally, the stability of a protein can be measured through several techniques, one such technique includes the controlled application of tension to proteins in an atomic force microscopy (AFM) framework.  This kind of tension-based approach is of interest as it probes the force required to unfold individual domains of a protein rather than a bulk characteristic like molting point or activity. It has been shown that key features observed in an AFM experiment can be well reproduced with molecular dynamics simulation, which has been applied to characterize the mechanisms of unfolding of proteins as well as ligand-protein interactions.  Steered molecular dynamics (SMD) was applied to pull and unfold proteins and determine the force required to unfold them. The relative force required to unfold different models with the same sequence was used to estimate relative model accuracy.  This follows from the hypothesis that the structural stability of a given model’s conformation would positively correlate with its accuracy, i.e. how close that model is to its native fold. It was found that near-native models could be successfully selected by comparing the forces required to unfold models, indicating that high unfolding forces indeed indicated high model stability, which in turn correlated with model accuracy. I also applied molecular dynamics-based approaches for refinement of protein structures that are determined from cryo-EM density maps. Computational approaches for protein structure refinement are often developed with the design aim of requiring a user input and experimental data. I modeled the atomic structure of the major capsid protein gp27 and the decoration protein gp26 of PhageG to a 6.1Å resolution electron microscopy map. PhageG modeling was done by mapping the sequences to a presumed homolog (Hk97), arranging the subunits into hexamers and trimmers as suggested by mass spectroscopy data, rigid docking to respective map segments, refinement against half maps using MDFF across a range of weights, and then finally refinement to the whole map using the optimized weight. I also modeled the atomic structure of the protein USP7 to an 8.2 Å resolution map. USP7 modeling was done by combining crystalized domains of the whole structure, rigidly docking the model to the EM map by hand, and then refining in a similar manner as PhageG, with the added approach of weight scaling to overcome local minima along the relaxation. The USP7 model was further validated by exhibiting a ligand-protein binding pose, determined by glide, which corresponded to enzymatic activity mutation assays. In summary I applied molecular dynamics, in conjunction with other computational methods, towards protein structure determination, refinement, and quality assessment.</p>

The B family of DNA-dependent DNA polymerases (pols) are uniquely sensitive to inhibition by N2-(p-n-butylphenyl)deoxyguanosine 5'-triphosphate (BuPdGTP). The affinity of members of the B family for BuPdGTP varies greatly, as does the ability of certain members to use the modified nucleotide as a substrate. For example, eukaryotic pol α has high affinity for the nucleotide, but incorporates it into DNA poorly, while T4 pol has lower affinity, but incorporates the nucleotide well. This thesis addresses two questions: 1) What are the amino acid residue(s) that impart sensitivity to BuPdGTP? and 2) is incorporation of BuPdGTP required for inhibition? To answer the first question, molecular modeling was used with the crystal structure of RB69 pol [Wang et al., 1997], an enzyme closely related to T4 pol [Wang et al., 1995]. This modeling identified a structural pocket adjacent to the polymerase active site that could serve as the butylphenyl "receptor". Based upon this modeling, a chimeric T4 pol containing the residues corresponding to the butylphenyl receptor from human pol α was designed and engineered for expression. The chimera was hypothesized to have a pol α-like phenotype with respect to its response to BuPdGTP (higher sensitivity/ lost ability to incorporate). The chimera was found to be unstable during purification, leaving the hypothesis unresolved. To answer the second question, non-substrate derivatives of BuPdGTP were in which the α,β anhydride oxygen of the triphosphate were replaced with either a CH2 or NH. The ability of the latter derivatives to inhibit polymerase activity and to serve as substrates was measured on T4 pol, RB69 pol and human pol α. Both derivatives retained high potency, but were not substrates under the conditions tested. These compounds were potent, selective inhibitors of B family pol that should be useful in the formation of a stable complex of enzyme:DNA:inhibitor for crystallization trials.

Many double-stranded DNA viruses use a packaging motor during maturation to recognize and transport genetic material into the capsid. In terminase motors, the TerS complex recognizes DNA, while the TerL motor packages the DNA into the capsid shell. Although there are several models for DNA recognition and translocation, how the motor components assemble and power DNA translocation is unknown. Using the thermophilic P74-26 bacteriophage model system, we discover that TerL uses a trans-activated ATP hydrolysis mechanism. Additionally, we identify critical residues for TerL ATP hydrolysis and DNA binding. With a combination of x-ray crystallography, SAXS, and molecular docking, we build a structural model for TerL pentamer assembly. Apo and ATP analog-bound TerL ATPase domain crystal structures show ligand-dependent conformational changes, which we propose power DNA translocation. Together, we assimilate these findings to build models for both motor assembly and DNA translocation. Additionally, with the P76-26 system, we identify the TerS protein as gp83. I find that P74-26 TerS is a nonameric ring that stimulates TerL ATPase activity while inhibiting TerL nuclease activity. Using cryoEM, I solve 3.8 Å and 4.8 Å resolution symmetric and asymmetric reconstructions of the TerS ring. I observe in P74-26 TerS, the conserved C-terminal beta-barrel is absent, and instead the region is flexible or unstructured. Furthermore, the helix-turn-helix motifs of P74-26 TerS are positioned differently than those of known TerS structures, suggesting P74-26 uses an alternative mechanism to recognize DNA.