Crystal structures of bacterial proteins, PenA and CagL, related to human pathogenicity

Abstract

i.Structural study of class A β-lactamase PenA in Burkholderia thailandenesis The β-lactamase, which was produced for protection to antibiotics in bacterium, generally reveals antibiotic resistance by hydrolyzing the antibiotics’β-lactam ring. Until present, Almost of bacterium using β-lactamase such as TEM and SHV evolved for survival against a variety of antibiotics through structural change by mutation such as deletion, substitutions, and repeat unit insertion of amino acid. Among β-lactamases, the Pen A used in this study belongs to class A β-lactamase in B. thailandenesis that it originally exhibits an antibiotic resistance to penicillin such as amoxicillin. In addition to the well-known β-lactamases including SHV-1 and TEM-1, PenA also was found not only change to ESBL by mutating such as substitution and deletion but also switching of two phases reversely by TR duplication, resulting that can increase resistance to third generation cephalosporin such as ceftazidime. Although study about point mutation for ESBL was already known from other β-lactamases, the studies about an accurate structural mechanism by insertion of long repetitive amino acid for inactivation of antibiotics are little. So, structures of PenA-WT and mutants including TR mutants (TR10 and TR11) and point mutants (N136D and C69Y) are investigated. Each protein are expressed, purified, and obtained x-ray crystallographic data at high resolution (1.4-1.7 Å) without and with ceftazidime-like glycyl boronic acid (CBA), respectively. Result of structure analysis, the major change between PenA-WT and mutants can show commonly change of omega (Ω) loop on active site through conformation change for the increased cavity size. This change increases not only binding affinity to antibiotic such as third generation cephalosporin, ceftazidime but also catalysis of hydrolysis activity. Consequently, change of omega loop at active site is very important role to change of substrate spectrum and induces the increase of resistance to a variety of antibiotic. Using these results, we can suggest that study to architecture of active site with CBA provides information to new drug development.

ii.Structural Study of CagL, a Component of the T4SS Pilus Assembly in H. pylori k74 strain. Type 4 secretion system (T4SS) in virulent H.pylori isolates, which are encoded with Cag (cytotoxic-associated gene) pathogenicity island, 40kb DNA, plays a very important role to interact with receptors of host cell and transfer CagA into host cell. CagL is characterized to interact with integrin through its flexible RGD domain during CagA translocation via a type 4 secretion system (T4SS) of Helicobacter pylori (Hp). Herein, to investigate interaction of pathogen-host cell, the CagL protein from H.pylori K74 strain was preferentially purified and was crystallized Se-Met and native CagL protein. Crystals of Se-Met and native CagL were subjected to x-ray diffraction at 2.9 Å and 2.8 Å resolution. It belonged to the same tetragonal space group P43212 and a dimer was found in asymmetry unit. Oligomeric state of CagL in solution was identified as monomer by AUC. Overall structure of monomer CagL reveals like a helix bundle consisting of four elongated α-helices and two α-short helices. The RGD motif, which is known to interact with integrin, is found in the long rigid α2 helix flanked by the conserved protease sensitive and RGD helper sequences. This indicates the possible unfolding of RGD region for integrin association. However, in comparison with 26695 CagL, the loop of CagL-K74 connecting α1 to α2 was well defined, and two additional salt bridges were found between the helices, suggesting that the putative flexible region harboring RGD motif might be more stable in this CagL variant. Also, because CagL is known to complex formation with CagI and CagH in cell, how CagL forms complex with other cag proteins such as CagI and CagH and how it interacts with host cell receptor integrin will be more study.