Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site

OmpT from Escherichia coli belongs to a family of highly homologous outer membrane proteases, known as omptins, which are implicated in the virulence of several pathogenic Gram‐negative bacteria. Here we present the crystal structure of OmpT, which shows a 10‐stranded antiparallel β‐barrel that protrudes far from the lipid bilayer into the extracellular space. We identified a putative binding site for lipopolysaccharide, a molecule that is essential for OmpT activity. The proteolytic site is located in a groove at the extracellular top of the vase‐shaped β‐barrel. Based on the constellation of active site residues, we propose a novel proteolytic mechanism, involving a His—Asp dyad and an Asp—Asp couple that activate a putative nucleophilic water molecule. The active site is fully conserved within the omptin family. Therefore, the structure described here provides a sound basis for the design of drugs against omptin‐mediated bacterial pathogenesis. Coordinates are in the Protein Data Bank (accession No. 1I78)

[1]  M. Egmond,et al.  Substrate specificity of the integral membrane protease OmpT determined by spatially addressed peptide libraries. , 2001, Biochemistry.

[2]  G. Rummel,et al.  Crystal structures explain functional properties of two E. coli porins , 1992, Nature.

[3]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[4]  E Harper,et al.  On the size of the active site in proteases: pronase. , 1972, Biochemical and biophysical research communications.

[5]  J. Rosenbusch,et al.  Structural basis for sugar translocation through maltoporin channels at 3.1 A resolution , 1995, Science.

[6]  T. Nishihara,et al.  Purification, characterization, and primary structure of Escherichia coli protease VII with specificity for paired basic residues: identity of protease VII and OmpT , 1988, Journal of bacteriology.

[7]  K. Diederichs,et al.  A conserved structural motif for lipopolysaccharide recognition by procaryotic and eucaryotic proteins. , 2000, Structure.

[8]  Luc Moulinier,et al.  Transmembrane Signaling across the Ligand-Gated FhuA Receptor Crystal Structures of Free and Ferrichrome-Bound States Reveal Allosteric Changes , 1998, Cell.

[9]  G. Schulz,et al.  The structure of the outer membrane protein OmpX from Escherichia coli reveals possible mechanisms of virulence. , 1999, Structure.

[10]  T. Quan,et al.  A surface protease and the invasive character of plague. , 1992, Science.

[11]  C. Eckerskorn,et al.  High-level biosynthetic substitution of methionine in proteins by its analogs 2-aminohexanoic acid, selenomethionine, telluromethionine and ethionine in Escherichia coli. , 1995, European journal of biochemistry.

[12]  G. Schulz,et al.  Structure of the outer membrane protein A transmembrane domain , 1998, Nature Structural Biology.

[13]  M. Lundrigan,et al.  Prevalence of ompT among Escherichia coli isolates of human origin. , 1992, FEMS microbiology letters.

[14]  L. Lally.  The CCP 4 Suite — Computer programs for protein crystallography , 1998 .

[15]  J. Deisenhofer,et al.  Crystal structure of the outer membrane active transporter FepA from Escherichia coli , 1999, Nature Structural Biology.

[16]  Colin Hughes,et al.  Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export , 2000, Nature.

[17]  G. Georgiou,et al.  Identification of OmpT as the Protease That Hydrolyzes the Antimicrobial Peptide Protamine before It Enters Growing Cells ofEscherichia coli , 1998, Journal of bacteriology.

[18]  M. Egmond,et al.  Identification of active site serine and histidine residues in Escherichia coli outer membrane protease OmpT , 2000, FEBS letters.

[19]  G. Schulz,et al.  Structure of the membrane channel porin from Rhodopseudomonas blastica at 2.0 Å resolution , 1994, Protein science : a publication of the Protein Society.

[20]  G. Schulz,et al.  The three‐dimensional structure of porin from Rhodobacter capsulatus at 3 Å resolution , 1990, FEBS letters.

[21]  J. Goguen,et al.  Nucleotide sequence of the plasminogen activator gene of Yersinia pestis: relationship to ompT of Escherichia coli and gene E of Salmonella typhimurium , 1989, Infection and immunity.

[22]  M. Egmond,et al.  In vitro folding, purification and characterization of Escherichia coli outer membrane protease ompT. , 2000, European journal of biochemistry.

[23]  K. H. Kalk,et al.  Structural evidence for dimerization-regulated activation of an integral membrane phospholipase. , 1999 .

[24]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[25]  K. Diederichs,et al.  Siderophore-mediated iron transport: crystal structure of FhuA with bound lipopolysaccharide. , 1998, Science.

[26]  M. Goldberg,et al.  Disruption of IcsP, the major Shigella protease that cleaves IcsA, accelerates actin‐based motility , 1997, Molecular microbiology.

[27]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[28]  Guan-qiao Yu,et al.  Identification and nucleotide sequence of the activator gene of the externally induced phosphoglycerate transport system of Salmonella typhimurium. , 1986, Gene.

[29]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

[30]  P. Sansonetti,et al.  SopA, the outer membrane protease responsible for polar localization of IcsA in Shigella flexneri , 1997, Molecular microbiology.

[31]  C. Stathopoulos.  Structural features, physiological roles, and biotechnological applications of the membrane proteases of the OmpT bacterial endopeptidase family: a micro-review. , 1998, Membrane & cell biology.

[32]  Y. Stierhof,et al.  New outer membrane-associated protease of Escherichia coli K-12 , 1994, Journal of bacteriology.

[33]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[34]  Neil D. Rawlings,et al.  [2] Families of serine peptidases , 1994, Methods in Enzymology.

[35]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[36]  R Miller,et al.  Optimizing Shake-and-Bake for proteins. , 1999, Acta crystallographica. Section D, Biological crystallography.

[37]  R M Esnouf,et al.  An extensively modified version of MolScript that includes greatly enhanced coloring capabilities. , 1997, Journal of molecular graphics & modelling.