Patrick Yip

Crystal structure of a class I α1,2‐mannosidase involved in N ‐glycan processing and endoplasmic reticulum quality control

Mannose trimming is not only essential for N‐glycan maturation in mammalian cells but also triggers degradation of misfolded glycoproteins. The crystal structure of the class I α1,2‐mannosidase that trims Man9GlcNAc2 to Man8GlcNAc2 isomer B in the endoplasmic reticulum of Saccharomyces cerevisiae reveals a novel (αα)7‐barrel in which an N‐glycan from one molecule extends into the barrel of an adjacent molecule, interacting with the essential acidic residues and calcium ion. The observed protein–carbohydrate interactions provide the first insight into the catalytic mechanism and specificity of this eukaryotic enzyme family and may be used to design inhibitors that prevent degradation of misfolded glycoproteins in genetic diseases.

AlgK is a TPR-containing protein and the periplasmic component of a novel exopolysaccharide secretin

The opportunistic pathogen Pseudomonas aeruginosa causes chronic biofilm infections in cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by overproduction of the exopolysaccharide alginate. Here we show that AlgK, a protein essential for production of high molecular weight alginate, is an outer membrane lipoprotein that contributes to the correct localization of the porin AlgE. Our 2.5 A structure shows AlgK is composed of 9.5 tetratricopeptide-like repeats, and three putative sites of protein-protein interaction have been identified. Bioinformatics analysis suggests that BcsA, PgaA, and PelB, involved in the production and export of cellulose, poly-beta-1,6-N-Acetyl-D-glucosamine, and Pel exopolysaccharide, respectively, share the same topology as AlgK/E. Together, our data suggest that AlgK plays a role in the assembly of the alginate biosynthetic complex and represents the periplasmic component of a new type of outer membrane secretin that differs from canonical bacterial capsular polysaccharide secretion systems.

Structure of Penicillium citrinum alpha 1,2-mannosidase reveals the basis for differences in specificity of the endoplasmic reticulum and Golgi class I enzymes.

Class I α1,2-mannosidases (glycosylhydrolase family 47) are key enzymes in the maturation of N-glycans. This protein family includes two distinct enzymatically active subgroups. Subgroup 1 includes the yeast and human endoplasmic reticulum (ER) α1,2-mannosidases that primarily trim Man9GlcNAc2 to Man8GlcNAc2 isomer B whereas subgroup 2 includes mammalian Golgi α1,2-mannosidases IA, IB, and IC that trim Man9GlcNAc2 to Man5GlcNAc2 via Man8GlcNAc2 isomers A and C. The structure of the catalytic domain of the subgroup 2 α1,2-mannosidase fromPenicillium citrinum has been determined by molecular replacement at 2.2-Å resolution. The fungal α1,2-mannosidase is an (αα)7-helix barrel, very similar to the subgroup 1 yeast (Vallée, F., Lipari, F., Yip, P., Sleno, B., Herscovics, A., and Howell, P. L. (2000) EMBO J. 19, 581–588) and human (Vallée, F., Karaveg, K., Herscovics, A., Moremen, K. W., and Howell, P. L. (2000) J. Biol. Chem. 275, 41287–41298) ER enzymes. The location of the conserved acidic residues of the catalytic site and the binding of the inhibitors, kifunensine and 1-deoxymannojirimycin, to the essential calcium ion are conserved in the fungal enzyme. However, there are major structural differences in the oligosaccharide binding site between the two α1,2-mannosidase subgroups. In the subgroup 1 enzymes, an arginine residue plays a critical role in stabilizing the oligosaccharide substrate. In the fungal α1,2-mannosidase this arginine is replaced by glycine. This replacement and other sequence variations result in a more spacious carbohydrate binding site. Modeling studies of interactions between the yeast, human and fungal enzymes with different Man8GlcNAc2 isomers indicate that there is a greater degree of freedom to bind the oligosaccharide in the active site of the fungal enzyme than in the yeast and human ER α1,2-mannosidases.

Dimeric c-di-GMP is Required for Post-translational Regulation of Alginate Production in Pseudomonas aeruginosa

Background: Alg44 regulates the production of alginate in Pseudomonas aeruginosa via c-di-GMP binding. Results: The structure of the PilZ domain of Alg44 in complex with c-di-GMP reveals residues that control c-di-GMP/Alg44 stoichiometry. Conclusion: Binding of dimeric c-di-GMP is required for alginate biosynthesis. Significance: This is the first example of a receptor requiring a specific form of c-di-GMP for activation. Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.

Combining in situ proteolysis and mass spectrometry to crystallize Escherichia coli PgaB

The periplasmic poly-β-1,6-N-acetyl-D-glucosamine (PNAG) de-N-acetylase PgaB from Escherichia coli was overexpressed and purified, but was recalcitrant to crystallization. Use of the in situ proteolysis technique produced crystals of PgaB, but these crystals could not be optimized for diffraction studies. By analyzing the initial crystal hits using SDS-PAGE and mass spectrometry, the boundaries of the protein species that crystallized were determined. The re-engineered protein target crystallized reproducibly without the addition of protease and with significantly increased crystal quality. Crystals of the selenomethionine-incorporated protein exhibited the symmetry of space group P2(1)2(1)2(1) and diffracted to 2.1 Å resolution.

Structures of 5-methylthioribose kinase reveal substrate specificity and unusual mode of nucleotide binding.

The methionine salvage pathway is ubiquitous in all organisms, but metabolic variations exist between bacteria and mammals. 5-Methylthioribose (MTR) kinase is a key enzyme in methionine salvage in bacteria and the absence of a mammalian homolog suggests that it is a good target for the design of novel antibiotics. The structures of the apo-form of Bacillus subtilis MTR kinase, as well as its ADP, ADP-PO4, AMPPCP, and AMPPCP-MTR complexes have been determined. MTR kinase has a bilobal eukaryotic protein kinase fold but exhibits a number of unique features. The protein lacks the DFG motif typically found at the beginning of the activation loop and instead coordinates magnesium via a DXE motif (Asp250-Glu252). In addition, the glycine-rich loop of the protein, analogous to the “Gly triad” in protein kinases, does not interact extensively with the nucleotide. The MTR substrate-binding site consists of Asp233 of the catalytic HGD motif, a novel twin arginine motif (Arg340/Arg341), and a semi-conserved W-loop, which appears to regulate MTR binding specificity. No lobe closure is observed for MTR kinase upon substrate binding. This is probably because the enzyme lacks the lobe closure/inducing interactions between the C-lobe of the protein and the ribosyl moiety of the nucleotide that are typically responsible for lobe closure in protein kinases. The current structures suggest that MTR kinase has a dissociative mechanism.

Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas fluorescens AlgK

AlgK is an outer-membrane lipoprotein involved in the biosynthesis of alginate in Pseudomonads and Azotobacter vinelandii. A recombinant form of Pseudomonas fluorescens AlgK with a C-terminal polyhistidine affinity tag has been expressed and purified from the periplasm of Escherichia coli cells and diffraction-quality crystals of AlgK have been grown using the hanging-drop vapour-diffusion method. The crystals grow as flat plates with unit-cell parameters a = 79.09, b = 107.85, c = 119.15 A, beta = 96.97 degrees. The crystals exhibit the symmetry of space group P2(1) and diffract to a minimum d-spacing of 2.5 A at Station X29 of the National Synchrotron Light Source, Brookhaven National Laboratory. On the basis of the Matthews coefficient (V(M) = 2.53 A3 Da(-1)), four protein molecules are estimated to be present in the asymmetric unit.

PelA and PelB proteins form a modification and secretion complex essential for Pel polysaccharide-dependent biofilm formation in Pseudomonas aeruginosa

The pellicle (PEL) polysaccharide is synthesized by the opportunistic pathogen Pseudomonas aeruginosa and is an important biofilm constituent critical for bacterial virulence and persistence. PEL is a cationic polymer that promotes cell–cell interactions within the biofilm matrix through electrostatic interactions with extracellular DNA. Translocation of PEL across the outer membrane is proposed to occur via PelB, a membrane-embedded porin with a large periplasmic domain predicted to contain 19 tetratricopeptide repeats (TPRs). TPR-containing domains are typically involved in protein–protein interactions, and we therefore sought to determine whether PelB serves as a periplasmic scaffold that recruits other components of the PEL secretion apparatus. In this study, we show that the TPR domain of PelB interacts with PelA, an enzyme with PEL deacetylase and hydrolase activities. Structure determination of PelB TPRs 8–11 enabled us to design systematic deletions of individual TPRs and revealed that repeats 9–14, which are required for the cellular localization of PelA with PelB are also essential for PEL-dependent biofilm formation. Copurification experiments indicated that the interaction between PelA and PelB is direct and that the deacetylase activity of PelA increases and its hydrolase activity decreases when these proteins interact. Combined, our results indicate that the TPR-containing domain of PelB localizes PelA to the PEL secretion apparatus within the periplasm and that this may allow for efficient deacetylation of PEL before its export from the cell.