Active-Site Gating Regulates Substrate Selectivity in a Chymotrypsin-Like Serine Protease

Abstract

We report here the first structure of a member of the IgA protease family at 1.75Å resolution. This protease is a founding member of the Type V (autotransporter) secretion system and is considered a virulence determinant among the bacteria expressing the enzyme. The structure of the enzyme fits that of a classical autotransporter in which several unique domains necessary for protein function are appended to a central, 100 Å long β-helical domain. The N-terminal domain of the IgA protease is found to possess a chymotrypsin-like fold. However, this catalytic domain contains a unique loop D that extends over the active site acting as a lid, gating substrate access. The data presented provide a structural basis for the known ability of IgA proteases to only cleave the P/S/T rich hinge peptide unique to IgA1 in the context of the intact fold of the immunoglobulin. Based upon the structural data as well as molecular modeling, a model is presented that suggests the unique, extended loop D in this IgA protease sterically occludes the active site binding cleft in the absence of immunoglobulin binding. Only in the context of binding of the IgA1 immunoglobulin Fc domain in a valley formed between the N-terminal protease domain and another domain appended to the β-helix spine (domain-2) is the lid stabilized in an open conformation. The stabilization of this open conformation through Fc association subsequently allows access of the hinge peptide to the active site resulting in recognition and cleavage of the substrate.

The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping

“…Despite their abundance and the increasing amount of data linking AT proteins to bacterial virulence, very little structural information is available at a molecular level for these proteins. In fact, AT proteins are significantly underrepresented in the Protein Data Bank (PDB), with only 1 fulllength AT structure, 7 AT passenger domains, 5 AT β-domains, and 12 small domains of trimeric autotransporters deposited among the ∼87,500 structures currently available in the PDB (9,18,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). None of these entries belong to the large family of AIDA-I-type AT proteins from Gamma-Proteobacteria (3,16).…”

A Conserved Aromatic Residue in the Autochaperone Domain of the Autotransporter Hbp Is Critical for Initiation of Outer Membrane Translocation

“…The side chain points inward in the ␤-helical lumen and stacks with other aromatic residues farther up in the stem. Sequence alignments of SPATEs and related serine protease ATs showed that a Trp residue is most often found at this position, although, for example, the Haemophilus influenzae ATs IgA protease (14) and Hap (46) have a Tyr and Phe at this position, respectively. ␤-Helix structures are usually capped both at the C terminus and the N terminus to prevent inappropriate interactions with the hydrophobic core and the unpaired strands (16).…”

“…Structural analysis of the secreted AT passengers pertactin (11), VacA (12), hemoglobin protease (Hbp) (13), and IgA protease (14) reveals a common overall topology: a long stem or stalk consisting of a right-handed ␤-helix to which other functional domains are appended. A ␤-helix is an extremely stable fold of ␤-strands, in this case of triangular rungs comprising three strands, that are interrupted by loops with variable length and are stacked to form a cross ␤-structure (15,16).…”

A Genome-Scale Proteomic Screen Identifies a Role for DnaK in Chaperoning of Polar Autotransporters inShigella

“…Then, in a coordinated fashion, the carboxy-terminal ␤-barrel is inserted into the outer membrane, and the passenger domain is translocated to the bacterial surface, although the mechanism by which these processes occur is poorly understood. At the bacterial surface, the passenger domains of all autotransporters for which the structures have been solved adopt a long, righthanded parallel ␤-helical conformation (20,22,37,52).…”