Conserved Water Molecules Stabilize the Ω-Loop in Class A β-Lactamases

Abstract

A set of 49 high-resolution (<2.2 Å) structures of the TEM, SHV, and CTX-M class A ␤-lactamase families was systematically analyzed to investigate the role of conserved water molecules in the stabilization of the ⍀-loop. Overall, 13 water molecules were found to be conserved in at least 45 structures, including two water positions which were found to be conserved in all structures. Of the 13 conserved water molecules, 6 are located at the ⍀-loop, forming a dense cluster with hydrogen bonds to residues at the ⍀-loop as well as to the rest of the protein. This layer of conserved water molecules is packed between the ⍀-loop and the rest of the protein and acts as structural glue, which could reduce the flexibility of the ⍀-loop. A correlation between conserved water molecules and conserved protein residues could in general not be detected, with the exception of the conserved water molecules at the ⍀-loop. Furthermore, the evolutionary relationship between the three families, derived from the number of conserved water molecules, is similar to the relationship derived from phylogenetic analysis.␤-Lactamases (EC 3.5.2.6) hydrolyze the ␤-lactam ring and therefore are the main cause for resistance against ␤-lactam antibiotics. A sequence-based classification scheme was initially proposed by Ambler (1) and soon thereafter was extended by others (14, 28). Class A ␤-lactamases include the plasmid-borne TEM, SHV, and CTX-M ␤-lactamases, which are found most commonly in gram-negative bacteria. The TEM and SHV ␤-lactamase families are closely related (with a sequence identity of 67%), while the CTX-M family is more distant (with a sequence identity of 40% to TEM and SHV) (36). The members of the TEM and SHV families differ by only one to seven amino acids, while the members of the CTX-M family are more diverse (80% sequence identity) (13). Because of their importance in antibiotic resistance, the enzymes of class A ␤-lactamases are well studied and characterized (2,6,10,20,21,30), and a vast amount of crystallographic data is available. While the sequence similarity between the protein families is moderate, the structures of all TEM, SHV, and CTX-M ␤-lactamases show a remarkably high similarity. The enzymes are composed of two domains that are closely packed together: an all-␣ domain, consisting of eight ␣-helices, and an ␣/␤ domain, consisting of three ␣-helices and five ␤-sheets (15). The active site cavity is part of the interface between the two domains and is limited by the ⍀-loop. The ⍀-loop (residues 161 to 179) is a conserved structural element in all class A ␤-lactamases and is directly involved in the catalytic reaction of the enzymes because it positions the general base Glu166. Its conformation is anchored by a highly conserved salt bridge formed between Arg164 and Asp179 (19,37,38). However, the long ⍀-loop has only a few contacts with the rest of the protein and was therefore speculated to be a flexible element (20). Indeed, a molecular dynamics study (32) attributes some flexibility to a part of the ⍀-loop. How...

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Molecular Mechanism of the Hydration of Candida antarctica Lipase B in the Gas Phase: Water Adsorption Isotherms and Molecular Dynamics Simulations

“…For water activities below a critical water activity (a w = 0.5 for CALB) water initially binds to well-defined water-binding sites at the protein surface, [25,31] and then gradually a spanning water network is formed. [33,39,50] The amount of protein-bound water increased linearly with water activity, depending on the number of water-binding sites on the surface and their affinity toward water.…”

“…[28] Comparative studies of crystal structures showed that there are water binding sites on the surface and on the interior of a protein, which are occupied by water molecules in different crystal structures. [29][30][31] Computer simulations of enzymes in different solvents are a valuable tool for investigating the effect of solvent on the structure and dynamics of proteins. [21,32,33] While in most simulations proteins showed no significant structural changes in different solvents, the molecular flexibility decreased when simulated in organic solvents, [33,34] which has been confirmed by different experimental techniques, such as time-resolved fluorescence anisotropy [35] and dielectric relaxation spectroscopy.…”

High resolution structural and functional analysis of a hemopexin motif protein from Dolichos

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