Oligomeric interactions provide alternatives to direct steric modes of control of sugar kinase/actin/hsp70 superfamily functions by heterotropic allosteric effectors: Inhibition of E. coli glycerol kinase

Abstract

Unlike those for monomeric superfamily members, heterotropic allosteric effectors of the tetrameric E. coli glycerol kinase (EGK) bind to only one of the two domains that define the catalytic cleft and far from the active site. An R369A amino acid substitution removes oligomeric interactions of a novel mini domain-swap loop of one subunit with the catalytic site of another subunit, and an A65T substitution perturbs oligomeric interactions in a second interface. Linked-functions enzyme kinetics, analytical ultracentrifugation, and FRET are used to assess effects of these substitutions on the allosteric control of catalysis. Inhibition by phosphotransferase system protein IIA Glc is reduced by the R369A substitution, and inhibition by fructose 1,6-bisphosphate is abolished by the A65T substitution. The oligomeric interactions enable the heterotropic allosteric effectors to act on both domains and modulate the catalytic cleft closure despite binding to only one domain.Its crystal structure [1] shows that EGK is a member of the sugar kinase/actin/hsp 70 superfamily of proteins [2]. 1 The common structure of superfamily members consists of two domains with the ATPase catalytic site located in a cleft between the domains, as shown in figure 1 for EGK. Catalysis is associated with relative movement of the domains that closes the cleft upon substrate binding [3,4]. The functional activities of several members of the superfamily, including EGK [5], hexokinases [6], actin [7], and hsp70 [8], are modulated by allosteric effectors, and it is generally believed that the effectors act on the cleft closure. For most superfamily members, crystal structures support this conclusion by showing that allosteric effectors interact with both domains. Allosteric effectors for actin [7] and glucokinase [9] as well as the peptide domain linker of hsp70 [10] bind to a hydrophobic cleft that is formed between the domains opposite the substrate binding sites. Nucleotide exchange factors for © 2009 Elsevier Inc. All rights reserved.*To whom correspondence should be addressed to Texas A&M University, Department of Biochemistry & Biophysics, 2128 TAMU, College Station, TX 77843-2128, dpettigrew@tamu.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 Abbreviations used: IIA Glc , the glucose-specific phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system (TC 4.A.1 (http://www.tcdb.org/)), also known as III glc and Crr; EGK, Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase); A65T EGK, EGK with the A65T amino acid substitution; R369A...

Mechanistic analysis of allosteric and non-allosteric effects arising from nanobody binding to two epitopes of the dihydrofolate reductase of Escherichia coli

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“…The GhACT17D ORF was 1134 bp in length and encoded a peptide containing 377 amino acid residues, comprising 38 strongly basic (+), 50 strongly acidic (−), 129 hydrophobic, and 86 polar amino acids. In a search against the NCBI protein sequence databases, the putative protein sequence of GhACT17D showed homology with actin proteins from other plants and contained the conserved nucleotide-binding domain (NBD) of the sugar kinase/HSP70/actin superfamily, which forms a deep cleft in which the nucleotide sits [ 26 ]. Eleven residues that compose this conserved feature were mapped to GhACT17D ( Figure 2 A).…”

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“…The allosteric function is then quantified as W ax = k 9 /k 3 )( 8 ) where k 3 and k 9 are as defined in Figure 1 B ( 7 , 8 ). Several V-type systems have been characterized using an allosteric energy cycle that includes changes in the rate of catalysis ( 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 ). A purely V-type response would be observed if Q ax = 1 and W ax ≠ 1.…”

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