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Christopher P Hill

Publications and source records attributed to Christopher P Hill.

10 recordsLinked to original sources
PubMed2003

The pore of activated 20S proteasomes has an ordered 7-fold symmetric conformation.

The 20S proteasome is a large multisubunit assembly that performs most of the intracellular non-lysosomal proteolysis of eukaryotes. Substrates access the proteasome active sites, which are sequestered in the interior of the barrel-shaped structure, through pores that are opened by binding of activator complexes. The crystal structure of yeast proteasome in complex with an 11S activator suggested that activation results from disordering of the proteasome gate residues. Here we report further analysis of this structure, which demonstrates that, in contrast to earlier models, the activated proteasome adopts an ordered 7-fold symmetric pore conformation that is stabilized by interactions formed by a cluster of highly conserved proteasome residues (Tyr8, Asp9, Pro17 and Tyr26). One non-canonical cluster, which appears to be mandated by the requirement that eukaryotic proteasomes also form an ordered closed conformation, explains all deviations from perfect conservation of these residues. We also demonstrate the importance of these conserved residues for proteolysis by an archaeal proteasome. Evolutionary considerations suggest that other activators might induce the same open proteasome conformation as seen with the 11S activator.

Amino Acid Sequence↗
PubMed2003

Structures along the catalytic pathway of PrmC/HemK, an N5-glutamine AdoMet-dependent methyltransferase.

Posttranslational methylation of release factors on the glutamine residue of a conserved GGQ motif is required for efficient termination of protein synthesis. This methylation is performed by an N(5)-glutamine methyltransferase called PrmC/HemK, whose crystal structure we report here at 2.2 A resolution. The electron density at the active site appears to contain a mixture of the substrates, S-adenosyl-L-methionine (AdoMet) and glutamine, and the products, S-adenosyl-L-homocysteine (AdoHcy) and N(5)-methylglutamine. The C-terminal domain of PrmC adopts the canonical AdoMet-dependent methyltransferase fold and shares structural similarity with the nucleotide N-methyltransferases in the active site, including use of a conserved (D/N)PPY motif to select and position the glutamine substrate. Residues of the PrmC (197)NPPY(200) motif form hydrogen bonds that position the planar Gln side chain such that the lone-pair electrons on the nitrogen nucleophile are oriented toward the methyl group of AdoMet. In the product complex, the methyl group remains pointing toward the sulfur, consistent with either an sp(3)-hybridized, positively charged Gln nitrogen, or a neutral sp(2)-hybridized nitrogen in a strained conformation. Due to steric overlap within the active site, proton loss and formation of the neutral planar methylamide product are likely to occur during or after product release. These structures, therefore, represent intermediates along the catalytic pathway of PrmC and show how the (D/N)PPY motif can be used to select a wide variety substrates.

Amino Acid Motifs↗
PubMed2003

Structure and ubiquitin binding of the ubiquitin-interacting motif.

Ubiquitylation is used to target proteins into a large number of different biological processes including proteasomal degradation, endocytosis, virus budding, and vacuolar protein sorting (Vps). Ubiquitylated proteins are typically recognized using one of several different conserved ubiquitin binding modules. Here, we report the crystal structure and ubiquitin binding properties of one such module, the ubiquitin-interacting motif (UIM). We found that UIM peptides from several proteins involved in endocytosis and vacuolar protein sorting including Hrs, Vps27p, Stam1, and Eps15 bound specifically, but with modest affinity (Kd = 0.1-1 mm), to free ubiquitin. Full affinity ubiquitin binding required the presence of conserved acidic patches at the N and C terminus of the UIM, as well as highly conserved central alanine and serine residues. NMR chemical shift perturbation mapping experiments demonstrated that all of these UIM peptides bind to the I44 surface of ubiquitin. The 1.45 A resolution crystal structure of the second yeast Vps27p UIM (Vps27p-2) revealed that the ubiquitin-interacting motif forms an amphipathic helix. Although Vps27p-2 is monomeric in solution, the motif unexpectedly crystallized as an antiparallel four-helix bundle, and the potential biological implications of UIM oligomerization are therefore discussed.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Structural insights into the catalytic mechanism of cyclophilin A.

Cyclophilins constitute a ubiquitous protein family whose functions include protein folding, transport and signaling. They possess both sequence-specific binding and proline cis-trans isomerase activities, as exemplified by the interaction between cyclophilin A (CypA) and the HIV-1 CA protein. Here, we report crystal structures of CypA in complex with HIV-1 CA protein variants that bind preferentially with the substrate proline residue in either the cis or the trans conformation. Cis- and trans-Pro substrates are accommodated within the enzyme active site by rearrangement of their N-terminal residues and with minimal distortions in the path of the main chain. CypA Arg55 guanidinium group probably facilitates catalysis by anchoring the substrate proline oxygen and stabilizing sp3 hybridization of the proline nitrogen in the transition state.

Amino Acid Sequence↗
PubMed2002

Crystal structure of the middle and C-terminal domains of the flagellar rotor protein FliG.

The FliG protein is essential for assembly, rotation and clockwise/counter-clockwise (CW/CCW) switching of the bacterial flagellum. About 25 copies of FliG are present in a large rotor-mounted assembly termed the 'switch complex', which also contains the proteins FliM and FliN. Mutational studies have identified the segments of FliG most crucial for flagellar assembly, rotation and switching. The structure of the C-terminal domain, which functions specifically in rotation, was reported previously. Here, we describe the crystal structure of a larger fragment of the FliG protein from Thermotoga maritima, which encompasses the middle and C-terminal parts of the protein (termed FliG-MC). The FliG-MC molecule consists of two compact globular domains, linked by an alpha-helix and an extended segment that contains a well-conserved Gly-Gly motif. Mutational studies indicate that FliM binds to both of the globular domains, and given the flexibility of the linking segment, FliM is likely to determine the relative orientation of the domains in the flagellum. We propose a model for the organization of FliG-MC molecules in the flagellum, and suggest that CW/CCW switching might occur by movement of the C-terminal domain relative to other parts of FliG, under the control of FliM.

Amino Acid Motifs↗
PubMed2002

Crystal structure of a biliverdin IXalpha reductase enzyme-cofactor complex.

Biliverdin reductase (BVR) catalyzes the last step in heme degradation by reducing the gamma-methene bridge of the open tetrapyrrole, biliverdin IXalpha, to bilirubin with the concomitant oxidation of a beta-nicotinamide adenine dinucleotide (NADH) or beta-nicotinamide adenine dinucleotide phosphate (NADPH) cofactor. Bilirubin is the major bile pigment in mammals and has antioxidant and anticompliment activity. We have determined X-ray crystal structures of apo rat BVR and its complex with NADH at 1.2 A and 1.5 A resolution, respectively. In agreement with an independent structure determination of the apo-enzyme, BVR consists of an N-terminal dinucleotide-binding domain (Rossmann-fold) and a C-terminal domain that contains a six-stranded beta-sheet that is flanked on one face by several alpha-helices. The C-terminal and N-terminal domains interact extensively, forming the active site cleft at their interface. The cofactor complex structure reported here reveals that the cofactor nicotinamide ring extends into the active site cleft, where it is adjacent to conserved amino acid residues and, consistent with the known stereochemistry of the reaction catalyzed by BVR, the si face of the ring is accessible for hydride transfer. The only titratable side-chain that appears to be suitably positioned to function as a general acid in catalysis is Tyr97. This residue, however, is not essential for catalysis, since the Tyr97Phe mutant protein retains 50% activity. This finding suggests that the dominant role in catalysis may be performed by hydride transfer from the cofactor, a process that may be promoted by proximity of the invariant residues Glu96, Glu123, and Glu126, to the nicotinamide ring.

Amino Acid Sequence↗
PubMed2002

The structure of Saccharomyces cerevisiae Met8p, a bifunctional dehydrogenase and ferrochelatase.

Sirohaem is a tetrapyrrole-derived prosthetic group that is required for the essential assimilation of sulfur and nitrogen into all living systems as part of the sulfite and nitrite reductase systems. The final two steps in the biosynthesis of sirohaem involve a beta-NAD(+)-dependent dehydrogenation of precorrin-2 to generate sirohydrochlorin followed by ferrochelation to yield sirohaem. In Saccharomyces cerevisiae, Met8p is a bifunctional enzyme that carries out both of these reactions. Here, we report the 2.2 A resolution crystal structure of Met8p, which adopts a novel fold that bears no resemblance to the previously determined structures of cobalt- or ferro-chelatases. Analysis of mutant proteins suggests that both catalytic activities share a single active site, and that Asp141 plays an essential role in both dehydrogenase and chelatase processes.

Amino Acid Sequence↗
PubMed2002

Structural basis of ubiquitylation.

The attachment of the small protein ubiquitin to other proteins, a process known as ubiquitylation, is a widespread form of post-translational modification that regulates numerous cellular functions in eukaryotes. Ubiquitylation is performed by complexes of E2 and E3 enzymes that are assembled and select substrates via a series of protein-protein interactions. Recent structure determinations of the ubiquitylation machinery have revealed some of the various protein-protein interfaces involved.

Ligases↗
PubMed2002

SUMO wrestling with specificity.

A new structure reveals how substrates are recognized for conjugation to the ubiquitin-like protein SUMO, a posttranslational modification that functions in activation of transcription factors, nuclear import, and cell division.

Binding Sites↗