Faculty Bibliography

DNA polymerases attach to the DNA sliding clamp through a common overlapping binding site. We identify a small-molecule compound that binds the protein-binding site in the Escherichia coli beta-clamp and differentially affects the activity of DNA polymerases II, III, and IV. To understand the molecular basis of this discrimination, the cocrystal structure of the chemical inhibitor is solved in complex with beta and is compared with the structures of Pol II, Pol III, and Pol IV peptides bound to beta. The analysis reveals that the small molecule localizes in a region of the clamp to which the DNA polymerases attach in different ways. The results suggest that the small molecule may be useful in the future to probe polymerase function with beta, and that the beta-clamp may represent an antibiotic target.

Uropathogenic Escherichia coli invade the urothelial umbrella cells by using the zipper mechanism. However, the details of the early events of this invasion, such as the formation of the phagocytic cup, are not yet well understood. We show here, using thin section electron microscopy and immunogold labeling, that the plasma membrane curves around the bacterial surface in the phagocytic cup. There exists a uniform gap between the bacterium and the urothelial membrane, and actin filaments are present in the phagocytic cup. We suggest that the action-reaction between the mechanical forces generated by pilus retraction of the bacterium and the actin polymerization in the urothelial cell plays a role in maintaining the phagocytic cup. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials

The agyria (lissencephaly)/pachygyria phenotypes are catastrophic developmental diseases characterized by abnormal folds on the surface of the brain and disorganized cortical layering. In addition to mutations in at least four genes-LIS1, DCX, ARX and RELN-mutations in a human alpha-tubulin gene, TUBA1A, have recently been identified that cause these diseases. Here, we show that one such mutation, R264C, leads to a diminished capacity of de novo tubulin heterodimer formation. We identify the mechanisms that contribute to this defect. First, there is a reduced efficiency whereby quasinative alpha-tubulin folding intermediates are generated via ATP-dependent interaction with the cytosolic chaperonin CCT. Second, there is a failure of CCT-generated folding intermediates to stably interact with TBCB, one of the five tubulin chaperones (TBCA-E) that participate in the pathway leading to the de novo assembly of the tubulin heterodimer. We describe the behavior of the R264C mutation in terms of its effect on the structural integrity of alpha-tubulin and its interaction with TBCB. In spite of its compromised folding efficiency, R264C molecules that do productively assemble into heterodimers are capable of copolymerizing into dynamic microtubules in vivo. The diminished production of TUBA1A tubulin in R264C individuals is consistent with haploinsufficiency as a cause of the disease phenotype

The structure of the E. coli beta clamp polymerase processivity factor has been solved in complex with primed DNA. Interestingly, the clamp directly binds the DNA duplex and also forms a crystal contact with the ssDNA template strand, which binds into the protein-binding pocket of the clamp. We demonstrate that these clamp-DNA interactions function in clamp loading, perhaps by inducing the ring to close around DNA. Clamp binding to template ssDNA may also serve to hold the clamp at a primed site after loading or during switching of multiple factors on the clamp. Remarkably, the DNA is highly tilted as it passes through the beta ring. The pronounced 22 degrees angle of DNA through beta may enable DNA to switch between multiple factors bound to a single clamp simply by alternating from one protomer of the ring to the other

The mammalian urothelium apical surface plays important roles in bladder physiology and diseases, and it provides a unique morphology for ultrastructural studies. Atomic force microscopy (AFM) is an emerging tool for studying the architecture and dynamic properties of biomolecular structures under near-physiological conditions. However, AFM imaging of soft tissues remains a challenge because of the lack of efficient methods for sample stabilization. Using a porous nitrocellulose membrane as the support, we were able to immobilize large pieces of soft mouse bladder tissue, thus enabling us to carry out the first AFM investigation of the mouse urothelial surface. The submicrometer-resolution AFM images revealed many details of the surface features, including the geometry of the urothelial plaques that cover the entire surface and the membrane interdigitation at the cell borders. This interdigitation creates a membrane zipper, likely contributing to the barrier function of the urothelium. In addition, we were able to image the intracellular bacterial communities of type 1-fimbriated bacteria grown between the intermediate filament bundles of the umbrella cells, shedding light on the bacterial colonization of the urothelium.

We have developed a new strategy to enrich and fractionate phosphopeptides from peptide mixtures based on the difference in their isoelectric points (pIs) after methyl esterification. After isoelectric focusing (IEF) of a methylated tryptic digest of a mixture of alpha-S-casein and beta-casein, phosphopeptides were selectively enriched at acidic and neutral pHs while nonphosphopeptides left the focusing gel because their pIs are higher than the upper limit of the immobilized pH gradient. We wrote a web-based program, pIMethylation, to predict the pIs for peptides with and without methyl esterification. Theoretical calculations using pIMethylation indicated that methylated phosphopeptides and non-phosphopeptides can be grouped on the basis of the number of phosphate groups and basic residues in each peptide. Our IEF results were consistent with theoretical pIs of methylated peptides calculated by pIMethylation. We also showed that 2,6-dihydroxy-acetophenone is superior to 2,5-dihydroxybenzoic acid as a matrix for MALDI Q-TOF MS of methylated phosphopeptides in both positive and negative ion modes

The surface of the mammalian urinary bladder is covered by a crystalline, asymmetric unit membrane (AUM) structure that contains the four major uroplakins (UPs): Ia, Ib, II and IIIa. UPIa and UPIb belong to the family of tetraspanins. Although UPIa and UPIb are structurally conserved, only UPIb could exit from the endoplasmic reticulum (ER) and reach the cell surface when expressed alone in 293T cells. Modifications of the large extracellular loop of UPIb, such as mutation of the N-glycosylation site or the cysteines involved in the formation of three disulfide bridges, or exchanging the large luminal loop of UPIb with that of UPIa did not affect the ability of UPIb to reach the cell surface. However, modifications of any of the four transmembrane domains of UPIb led to ER retention, suggesting that the proper formation of helical bundles consisting of the tetraspanin transmembrane domains is a prerequisite for UPIb to exit from the ER. Results of sedimentation analysis suggested that aggregate formation is a mechanism for ER retention

Protein misfolding caused by exposure to arsenite is associated with transcriptional activation of the AIRAP gene. We report here that AIRAP is an arsenite-inducible subunit of the proteasome's 19S cap that binds near PSMD2 at the 19S base. Compared to the wild-type, knockout mouse cells or C. elegans lacking AIRAP accumulate more polyubiquitylated proteins and exhibit higher levels of stress when exposed to arsenite, and proteasomes isolated from arsenite-treated AIRAP knockout cells are relatively impaired in substrate degradation in vitro. AIRAP's association with the 19S cap reverses the stabilizing affect of ATP on the 26S proteasome during particle purification, and AIRAP-containing proteasomes, though constituted of 19S and 20S subunits, acquire features of hybrid proteasomes with both 19S and 11S regulatory caps. These features include enhanced cleavage of peptide substrates and suggest that AIRAP adapts the cell's core protein degradation machinery to counteract proteotoxicity induced by an environmental toxin

Tetraspanin uroplakins (UPs) Ia and Ib, together with their single-spanning transmembrane protein partners UP II and IIIa, form a unique crystalline 2D array of 16-nm particles covering almost the entire urothelial surface. A 6 A-resolution cryo-EM structure of the UP particle revealed that the UP tetraspanins have a rod-shaped structure consisting of four closely packed transmembrane helices that extend into the extracellular loops, capped by a disulfide-stabilized head domain. The UP tetraspanins form the primary complexes with their partners through tight interactions of the transmembrane domains as well as the extracellular domains, so that the head domains of their tall partners can bridge each other at the top of the heterotetramer. The secondary interactions between the primary complexes and the tertiary interaction between the 16-nm particles contribute to the formation of the UP tetraspanin network. The rod-shaped tetraspanin structure allows it to serve as stable pilings in the lipid sea, ideal for docking partner proteins to form structural/signaling networks

Although it has been shown that mouse uroplakin (UP) Ia, a major glycoprotein of urothelial apical surface, can serve as the receptor for the FimH lectin adhesin of type 1-fimbriated Escherichia coli, the organism that causes a great majority of urinary tract infections, the glycan structure of this native receptor was unknown. Using a sensitive approach that combines in-gel glycosidase and protease digestions, permethylation of released glycans, and mass spectrometry, we have elucidated for the first time the native glycoform structures of the mouse UPIa receptor and those of its non-binding homolog, UPIb, and have determined the glycosylation site occupancy. UPIa presents a high level of terminally exposed mannose residues (located on Man(6)GlcNAc(2) to Man(9)GlcNAc(2)) that are capable of specifically interacting with FimH. We have shown that this property is conserved not only in the mouse uroplakins but also in cattle and, even more importantly, in human UPIa, thus establishing the concept that UPIa is a major urothelial receptor in humans and other mammals for the mannose-specific FimH variant. In contrast, our results indicate that most terminally exposed glycans of mouse UPIb are non-mannose residues, thus explaining the failure of FimH to bind to this UPIb. In cattle, on the other hand, complex carbohydrates constituted only about 20% of the UPIb N-linked glycans. Human UPIa contained exclusively high mannose glycans, and human UPIb contained only complex glycans. The drastically different carbohydrate processing of the UPIa and UPIb proteins, two closely related members of the tetraspanin family, may reflect differences in their folding and masking due to their interactions with their associated proteins, UPII and UPIIIa, respectively. Results from this study shed light on the molecular pathogenesis of urinary tract infections and may aid in the design of glyco-mimetic inhibitors for preventing and treating this disease