<p>Lipopolysaccharide, the physical barrier that protects Gram-negative bacteria from various antibiotics and environmental stressors, is anchored to the outer membrane by the phosphorylated, acylated disaccharide of glucosamine known as lipid A. Besides being necessary for the viability of most Gram-negative bacteria, lipid A interacts directly with specific mammalian immune cell receptors, causing an inflammatory response that can result in septic shock. The lipid A biosynthetic pathway contains nine enzymatic steps, the sixth being the phosphorylation of the tetraacyldisaccharide-1-phosphate (DSMP) precursor to form lipid IV_A by the inner membrane-bound kinase LpxK, a divergent member of the P-loop containing nucleotide triphosphate hydrolase superfamily. LpxK is the only known P-loop kinase to act on a lipid at the membrane interface.</p><p> We report herein multiple crystal structures of <italic>Aquifex aeolicus</italic> LpxK in apo as well as ATP, ADP/Mg<super>2+</super>, AMP-PCP, and chloride-bound forms. LpxK consists of two &alpha;/&beta;/&alpha; sandwich domains connected by a two-stranded &beta;-sheet linker. The N-terminal domain, which has most structural homology to other P-loop kinase family members, is responsible for catalysis at the P-loop and positioning of the DSMP substrate for phosphoryl transfer on the inner membrane. The smaller C-terminal domain, a substructure unique to LpxK, helps bind the nucleotide substrate using a 25º hinge motion about its base which also assembles the necessary catalytic residues at the active site.</p><p> Using a thin-layer chromatography-based radioassay, we have performed extensive kinetic characterization of the enzyme and demonstrate that LpxK activity <italic>in vitro</italic> is dependent on the presence of detergent micelles, the use of divalent cations, and formation of a ternary LpxK-ATP/Mg<super>2+</super>-DSMP complex. Implementing steady-state kinetic analysis of multiple point mutants, we identify crucial active site residues. We propose that the interaction of D99 with H261 acts to increase the pK_a of the imidazole group, which in turn serves as the catalytic base to deprotonate the 4&rsquo;-hydroxyl of DSMP. An analogous mechanism has not yet been reported for any member of the P-loop kinase family.</p><p> The membrane/lipid binding characteristics of LpxK have also been also investigated through a crystal structure of the LpxK-lipid IV_A product complex along with point mutagenesis of residues in the DSMP binding pocket. Critical contacts with the bound lipid include interactions along the glucosamine backbone and the 1-position phosphate group, especially through R171. Furthermore, analysis of truncation mutants of the N-terminal helix of LpxK demonstrates that this substructure is a critical hydrophobic contact point with the membrane, and that both charge-charge and hydrophobic interactions contribute to the localization of LpxK at the lipid bilayer. </p><p> Overall, this work has contributed significantly to the limited knowledge surrounding membrane-bound enzymes that act upon lipid substrates. It has also provided insight into the process of enzyme evolution as LpxK, while containing a similar core domain as other P-loop kinases, has developed multiple subdomains required for both cellular localization and recognition of novel substrates. Finally, the presence of multiple crystal structures and detailed understanding of the LpxK catalytic mechanism will improve the chances of successfully targeting this essential step in lipid A biosynthesis in the pursuit of novel antimicrobials.</p> / Dissertation

The fundamental process by which an alpha-helical membrane protein attains its ultimate structure has previously been depicted as two energetically distinct stages where (1) the transmembrane (TM) segments are first threaded into the membrane bilayer as stable alpha-helices; and then (2) laterally interact to form the correct tertiary and/or quaternary structures. Central to the second stage of this model is the presence of amino acid sequence motifs in the TM segments that provide interaction-compatible surfaces through which the TM alpha-helices interact. Although these ideas have proven to be pivotal to the progress of the membrane protein folding field, a growing number of examples indicates that a variety of additional factors work together to dictate the ultimate interaction fate of TM embedded segments. In this context, we expand on these factors and explore other properties that can modulate the association of TM alpha-helices. A peptide model of myelin proteolipid protein (PLP) TM4 is capable of TM helix-helix interactions in SDS and biological membranes. Increasing the side chain volumes of two disease relevant residues (Ala242 and A248) reduces peptide self-association, indicating that these sites mediate TM helix packing through van der Waals interactions. Examination of the PLP TM2 alpha-helix shows that it is also capable of self-association and that its dimeric state depends on the presence or absence of residues at its C-terminus. Specifically, this sensitivity was attributed to changes in local hydrophobicity; a decrease in hydrophobicity likely reduces detergent-peptide interactions, which disrupts peptide alpha-helicity and the effectiveness of a nearby interaction compatible surface. We take advantage of this finding to determine the feasibility of coupling helix-helix interactions to an external factor such as pH. Our results indicate that pH can indeed modulate the dimerization state of the TM2 peptide and does so through the change in protonation state of Glu88. Increasing our knowledge of the variables contributing to TM helix-helix interactions provides valuable insights into membrane protein folding and how mutations can compromise this process. This knowledge will allow us to expand our arsenal of approaches to counter membrane protein misassembly--and ultimately human disease.

The fundamental process by which an alpha-helical membrane protein attains its ultimate structure has previously been depicted as two energetically distinct stages where (1) the transmembrane (TM) segments are first threaded into the membrane bilayer as stable alpha-helices; and then (2) laterally interact to form the correct tertiary and/or quaternary structures. Central to the second stage of this model is the presence of amino acid sequence motifs in the TM segments that provide interaction-compatible surfaces through which the TM alpha-helices interact. Although these ideas have proven to be pivotal to the progress of the membrane protein folding field, a growing number of examples indicates that a variety of additional factors work together to dictate the ultimate interaction fate of TM embedded segments. In this context, we expand on these factors and explore other properties that can modulate the association of TM alpha-helices. A peptide model of myelin proteolipid protein (PLP) TM4 is capable of TM helix-helix interactions in SDS and biological membranes. Increasing the side chain volumes of two disease relevant residues (Ala242 and A248) reduces peptide self-association, indicating that these sites mediate TM helix packing through van der Waals interactions. Examination of the PLP TM2 alpha-helix shows that it is also capable of self-association and that its dimeric state depends on the presence or absence of residues at its C-terminus. Specifically, this sensitivity was attributed to changes in local hydrophobicity; a decrease in hydrophobicity likely reduces detergent-peptide interactions, which disrupts peptide alpha-helicity and the effectiveness of a nearby interaction compatible surface. We take advantage of this finding to determine the feasibility of coupling helix-helix interactions to an external factor such as pH. Our results indicate that pH can indeed modulate the dimerization state of the TM2 peptide and does so through the change in protonation state of Glu88. Increasing our knowledge of the variables contributing to TM helix-helix interactions provides valuable insights into membrane protein folding and how mutations can compromise this process. This knowledge will allow us to expand our arsenal of approaches to counter membrane protein misassembly--and ultimately human disease.

Cystic fibrosis is caused primarily by deletion of Phe508. An exciting discovery was that CFTR’s sister protein, the P-glycoprotein (P-gp) containing the equivalent mutation (ΔY490), could be repaired by a drug-rescue approach. Drug substrates showed specificity, and their mechanism involves direct binding to the transmembrane domains (TMDs) since arginine suppressor mutations were identified in TMDs that mimicked drug-rescue to promote maturation. We tested the possibility of rescuing CFTR processing mutants with a drug-rescue approach. 1) Arginine mutagenesis was performed on TM6, 8, and 12. 2) Correctors were tested for specificity. 3) Truncation mutants were used to map the VX-809 rescue site. Correctors 5a, 5c, and VX-809 were specific for CFTR. VX-809 appeared to specifically rescue CFTR by stabilizing TMD1. Therefore, the TMDs are potential targets to rescue CFTR. Rescue of P-gp and CFTR appeared to occur by different mechanisms since no arginine suppressor mutations were identified in CFTR.

Cystic fibrosis is caused primarily by deletion of Phe508. An exciting discovery was that CFTR’s sister protein, the P-glycoprotein (P-gp) containing the equivalent mutation (ΔY490), could be repaired by a drug-rescue approach. Drug substrates showed specificity, and their mechanism involves direct binding to the transmembrane domains (TMDs) since arginine suppressor mutations were identified in TMDs that mimicked drug-rescue to promote maturation. We tested the possibility of rescuing CFTR processing mutants with a drug-rescue approach. 1) Arginine mutagenesis was performed on TM6, 8, and 12. 2) Correctors were tested for specificity. 3) Truncation mutants were used to map the VX-809 rescue site. Correctors 5a, 5c, and VX-809 were specific for CFTR. VX-809 appeared to specifically rescue CFTR by stabilizing TMD1. Therefore, the TMDs are potential targets to rescue CFTR. Rescue of P-gp and CFTR appeared to occur by different mechanisms since no arginine suppressor mutations were identified in CFTR.

Mesoporous silica materials are versatile platforms for biological catalysis, isolation of small molecules for detection and separation applications. The design of mesoporous silica supports for tailored protein and biomolecule interactions has been limited by the techniques to demonstrate biomolecule location and functionality as a function of pore size. This work examines the interaction of proteins and lipid bilayers with engineered porous silica surfaces using spherical silica particles with tunable pore diameters (3 – 12 nm) in the range relevant to biomolecule uptake in the pores, and large particle sizes (5 - 15 µm) amenable to microscopy imaging The differentiation of protein location between the external surface and within the pore, important to applications requiring protein protection or catalytic activity in pores, is demonstrated. A protease / fluorescent protein system is used to investigate protein location and protection as a function of pore size, indicating a narrow pore size range capable of protein protection, slightly larger than the protein of interest and approaching the protease dimensions. Selective functionalization, in this case exterior-only surface functionalization of mesoporous particles with amines, is extended to larger pore silica materials. A reaction time dependent functionalization approach is demonstrated as the first visually confirmed, selective amine functionalization method in protein accessible supports. Mesoporous silica nanoparticles are effective supports for lipid bilayer membranes and membrane associated proteins for separations and therapeutic delivery, although the role of support porosity on membrane fluidity is unknown. Transport properties of bilayers in lipid filled nanoparticles as a function of pore diameter and location in the particle are measured for the first time. Bilayer diffusivity increases with increasing pore size and is independent of bilayer location within the core, mid or cap of the particle, suggesting uniform long range bilayer mobility in lipid filled pores. Application of lipid bilayers on mesoporous silica was examined for membrane associated proteins A unique method to adhere functional proteins in lipid bilayers on mesoporous silica particles is established using vesicles derived from cell plasma membranes and their associated proteins. This method of membrane protein investigation retains proteins within native lipid membranes, stabilizing proteins for investigation on supports.

No description available.

Syphilis is a global public health concern with 36.4 million cases worldwide and 11 million new infections per year. It is a chronic multistage disease caused by the spirochete bacterium Treponema pallidum and is transmitted by sexual contact, direct contact with lesions or vertically from an infected mother to her fetus. T. pallidum is a highly invasive pathogen that rapidly penetrates tight junctions of endothelial cells and disseminates rapidly via the bloodstream to establish widespread infection. Previous investigations conducted in our laboratory identified the surface-exposed adhesin, pallilysin, as a metalloprotease that degrades the host components laminin (major component of the basement membrane lining blood vessels) and fibrinogen (primary component of the coagulation cascade), as well as fibrin clots (function to entrap bacteria and prevent disseminated infection). Furthermore, pallilysin expressed on the surface of the non-invasive spirochete Treponema phagedenis conferred upon this bacterium the ability to degrade fibrin clots. It was hypothesized that pallilysin is integral to the process of T. pallidum dissemination, and interference with its functioning will prevent spread throughout the host and establishment of chronic infection. To test this hypothesis, a two-pronged approach was undertaken during my thesis research. Bioinformatics analyses were used to trace the evolutionary history of pallilysin in an attempt to gain further insight into its role in the pathogenesis of T. pallidum. The sequence conservation of pallilysin was analyzed in the context of its homologues. The bioinformatics analyses revealed homologues in three spirochete genera, namely Treponema, Spirochaeta, and Borrelia, presented in decreasing order of the degree of sequence conservation. The HEXXH motif, part of the active site of the pallilysin metalloprotease, was fully conserved only in T. pallidum and T. paraluiscuniculi, both of which are systemic pathogens. However, the flanking sequences showed a high degree of conservation, especially in the Treponema and Spirochaeta genera. The minimum laminin-binding region of pallilysin identified previously was partially conserved among the treponema and spirochaeta homologues with the highest degree of conservation observed with the homologues from T. paraluiscuniculi and T. phagedenis, as well as among the homologues from the human oral pathogens. In vitro dissemination studies were performed to investigate the dissemination capacity of T. phagedenis heterologously expressing pallilysin. Human Umbilical Vein Endothelial Cells were seeded and grown to confluence on permeable inserts coated with growth factor-reduced Matrigel to create an artificial endothelial barrier. Wild type T. phagedenis, and T. phagedenis transformed either with the pallilysin open reading frame or its empty shuttle vector, were incubated with the barriers under anaerobic conditions. Dissemination across the barrier was assessed as percent traversal by both dark-field microscopic counts of treponemes and real-time quantitative PCR of genomic DNA extracted from the treponemes. The results were inconclusive. However, a traversal trend suggested heterologous expression of pallilysin may facilitate traversal of T. phagedenis across the artificial endothelial barrier. This study presented the first step towards elucidating the role of pallilysin in endothelial monolayer traversal and provided supporting evidence for the role of pallilysin in the widespread dissemination of T. pallidum in vivo. / Graduate

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

No description available.