Faculty Bibliography

Gfh1, a transcription factor from Thermus thermophilus, inhibits all catalytic activities of RNA polymerase (RNAP). We characterized the Gfh1 structure, function and possible mechanism of action and regulation. Gfh1 inhibits RNAP by competing with NTPs for coordinating the active site Mg2+ ion. This coordination requires at least two aspartates at the tip of the Gfh1 N-terminal coiled-coil domain (NTD). The overall structure of Gfh1 is similar to that of the Escherichia coli transcript cleavage factor GreA, except for the flipped orientation of the C-terminal domain (CTD). We show that depending on pH, Gfh1-CTD exists in two alternative orientations. At pH above 7, it assumes an inactive 'flipped' orientation seen in the structure, which prevents Gfh1 from binding to RNAP. At lower pH, Gfh1-CTD switches to an active 'Gre-like' orientation, which enables Gfh1 to bind to and inhibit RNAP.

AMPA-type glutamate receptors (AMPARs) play a major role in excitatory synaptic transmission and plasticity. Channel properties are largely dictated by their composition of the four subunits, GluR1-4 (or A-D). Here we show that AMPAR assembly and subunit stoichiometry are determined by RNA editing in the pore loop. We demonstrate that editing at the GluR2 Q/R site regulates AMPAR assembly at the step of tetramerization. Specifically, edited R subunits are largely unassembled and ER retained, whereas unedited Q subunits readily tetramerize and traffic to synapses. This assembly mechanism restricts the number of the functionally critical R subunits in AMPAR tetramers. Therefore, a single amino acid residue affects channel composition and, in turn, controls ion conduction through the majority of AMPARs in the brain

The apical surface of terminally differentiated mammalian urothelial umbrella cells is covered by numerous plaques consisting of two-dimensional (2D) crystals of hexagonally packed 16 nm uroplakin particles, and functions as a remarkable permeability barrier. To determine the structural basis of this barrier function, we generated, by electron cryo microscopy, a projection map of the isolated mouse urothelial plaques at 7 A and a 3D structure at 10 A resolution. Our results indicate that each 16 nm particle has a central 6 nm lipid-filled 'hole' surrounded by 6 inverted U-shaped subunits, each consisting of an inner and an outer subdomain connected via a distal joint. The transmembrane portion of each subdomain can fit about 5 helices. This finding, coupled with our STEM and EM data, suggests that uroplakin pairs Ia/II and Ib/III are associated with the inner and outer subdomains, respectively. Since the inner subdomains interconnect to form a ring, which can potentially segregate the lipids of the central hole from those outside, the 2D crystalline uroplakin network may impose an organized state and a severely restricted freedom of movement on the lipid components, thus reducing membrane fluidity and contributing to the barrier function of urothelial plaques. Our finding that distinct uroplakin substructures are in contact with the cytoplasmic and exoplasmic leaflets of the plaque suggests that the two leaflets may have different lipid composition and contribute asymmetrically to the barrier function. We propose that the crystalline lattice structure of uroplakin, through its interactions with specialized lipids, plays a major role in the remarkable permeability barrier function of urothelial apical surface. Our results also have implications for the transmembrane signal transduction in urothelial cells as induced by the binding of uropathogenic E. coli to its uroplakin receptor

AMPA receptors are tetrameric cation channels that mediate the majority of fast excitatory transmission in the brain. Four subunits, GluR1-4 (or A-D) assemble in various stoichiometries, resulting in a spectrum of functionally distinct channels. Rules that govern assembly are largely unknown. The majority of AMPARs contain GluR2, which dominates transmission properties via Arg607, introduced into the pore loop by RNA editing (at the Q/R-site). Here we report that Arg607 also determines AMPAR assembly. Sedimentation analysis reveals that edited GluR2-R channels remain incompletely assembled and ER-retained, whereas unedited GluR2-Q channels readily tetramerize and exit from the ER, in neurons and HeLa cells. Mutagenesis reveals that the structure of the pore loop affects tetramerization. Therefore, by modulating a critical assembly surface, Q/R-editing determines AMPAR assembly and subunit stoichiometry

The binding of uropathogenic Escherichia coli to the urothelial surface is a critical initial event for establishing urinary tract infection, because it prevents the bacteria from being removed by micturition and it triggers bacterial invasion as well as host cell defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium and its urothelial receptor, uroplakin Ia (UPIa). To localize the UPIa receptor on the 16 nm particles that form two-dimensional crystals of asymmetric unit membrane (AUM) covering >90 % of the apical urothelial surface, we constructed a 15 A resolution 3-D model of the mouse 16 nm AUM particle by negative staining and electron crystallography. Similar to previous lower-resolution models of bovine and pig AUM particles, the mouse 16 nm AUM particle consists of six inner and six outer domains that are interconnected to form a twisted ribbon-like structure. Treatment of urothelial plaques with 0.02-0.1 % (v/v) Triton X-100 allowed the stain to penetrate into the membrane, revealing parts of the uroplakin transmembrane moiety with an overall diameter of 14 nm, which was much bigger than the 11 nm value determined earlier by quick-freeze deep-etch. Atomic force microscopy of native, unfixed mouse and bovine urothelial plaques confirmed the overall structure of the luminal 16 nm AUM particle that was raised by 6.5 nm above the luminal membrane surface and, in addition, revealed a circular, 0.5 nm high, cytoplasmic protrusion of approximately 14 nm diameter. Finally, a difference map calculated from the mouse urothelial plaque images collected in the presence and absence of recombinant bacterial FimH/FimC complex revealed the selective binding of FimH to the six inner domains of the 16 nm AUM particle. These results indicate that the 16 nm AUM particle is anchored by a approximately 14 nm diameter transmembrane stalk, and suggest that bacterial binding to UPIa that resides within the six inner domains of the 16 nm AUM particle may preferentially trigger transmembrane signaling involved in bacterial invasion and host cell defense

The binding of uropathogenic Escherichia coli to the urothelial surface is a crucial initial event for establishing urinary tract infection because it allows the bacteria to gain a foothold on the urothelial surface, thus preventing them from being removed by micturition. In addition, it triggers bacterial invasion as well as host urothelial defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium, a filamentous attachment apparatus, and its urothelial receptor. We have prepared a biotinylated, recombinant FimH-FimC adhesin:chaperone complex and used it to identify its mouse urothelial receptor. The FimH-FimC complex binds specifically to a single 24 kDa major mouse urothelial plaque protein, which we identified as uroplakin Ia by mass spectrometry, cDNA cloning and immunoreactivity. The terminal mannosyl moieties on Asn-169 of uroplakin Ia are responsible for FimH as well as concanavalin A binding. Although FimH binds to uroplakin Ia with only moderate strength (K(d) approximately 100 nM between pH 4 and 9), the binding between multiple fimbriae of a bacterium and the crystalline array of polymerized uroplakin receptors should achieve high avidity and stable bacterial attachment. The FimH-FimC complex binds preferentially to the mouse urothelial umbrella cells in a pattern similar to uroplakin staining. Our results indicate that the structurally related uroplakins Ia and Ib are glycosylated differently, that uroplakin Ia serves as the urothelial receptor for the type 1-fimbriated E. coli, and that the binding of uropathogenic bacteria to uroplakin Ia may play a key role in mediating the urothelial responses to bacterial attachment

The apical surfaces of urothelial cells are almost entirely covered with plaques consisting of crystalline, hexagonal arrays of 16 nm uroplakin particles. Although all four uroplakins, when SDS-denatured, can be digested by chymotrypsin, most uroplakin domains in native urothelial plaques are resistant to the enzyme, suggesting a tightly packed structure. The only exception is the C-terminal, cytoplasmic tail of UPIII (UPIII) which is highly susceptible to proteolysis, suggesting a loose configuration. When uroplakins are solubilized with 2% octylglucoside and fractionated with ion exchangers, UPIa and UPII were bound as a complex by a cation exchanger, whereas UPIb and UPIII were bound by an anion exchanger. This result is consistent with the fact that UPIa and UPIb are cross-linked to UPII and UPIII, respectively, and suggests that the four uroplakins form two pairs consisting of UPIa/II and UPIb/III. Immunogold labelling using a new mouse monoclonal antibody, AU1, revealed that UPIII is present in all urothelial plaques, indicating that the two uroplakin pairs are not segregated into two different types of urothelial plaque and that all plaques must have a similar uroplakin composition. Taken together, these results indicate that uroplakins form a tightly packed structure, that the four uroplakins interact specifically forming two pairs, and that both uroplakin pairs are required for normal urothelial plaque formation

Stem cell factor (SCF) plays important roles in hematopoiesis and the survival, proliferation, and differentiation of mast cells, melanocytes, and germ cells. SCF mediates its biological effects by binding to and activating a receptor tyrosine kinase designated c-kit or SCF receptor. In this report we describe the 2.3-A crystal structure of the functional core of recombinant human SCF. SCF is a noncovalent homodimer composed of two slightly wedged protomers. Each SCF protomer exhibits an antiparallel four-helix bundle fold. Dimerization is mediated by extensive polar and nonpolar interactions between the two protomers with a large buried surface area. Finally, we have identified a hydrophobic crevice and a charged region at the tail of each protomer that functions as a potential receptor-binding site. On the basis of these observations, a model for SCF small middle dotc-kit complex formation and dimerization is proposed

The B7: CD28/CTLA4 interaction plays a major role in T cell responses. Immune intervention targeted at this interaction has demonstrated a vast potential in enhancing tumor immunity and blocking autoimmunity and transplant rejection. However, the structural basis for this interaction is unclear. While we and others have performed site-directed mutagenesis to define amino acids involved in binding CD28 and CTLA4, these residues are localized in different regions, and it is unlikely for all of them to be directly involved. In addition, the effect of the mutations on the overall conformation of B7 has not been systematically evaluated. In this study, we have carried out site-directed mutagenesis to define the amino acids within B7-1 IgV-like domain which participate B7:CD28/CTLA4 interaction. Four anti-B7-1 mAbs that recognize three independent antigenic epitopes on B7-1 were used to monitor the effect of mutations on the overall conformation of B7-1. Of the five mutations in the IgV domain that we have produced, D113 > A appears to interfere with cell surface expression and/or overall conformation of B7-1. while four others do not significantly affect the overall conformation and cell surface expression of B7-1. Among them, G115 > A and Y91 > A eliminated B7-1 binding to both CD28Ig and CTLA4Ig; our previously reported mutants L109 > A and W88 > A selectively affect the B7-1 binding to either CD28Ig or CTLA4Ig. Structural modeling of B7-1 based on the structure of immunoglobulin revealed that these four and other previously identified critical amino acids in both IgV- and IgC-like domains can form a localized structure