IIA^Glc Inhibition of Glycerol Kinase:  A Communications Network Tunes Protein Motions at the Allosteric Site

Abstract

Steady-state and time-resolved fluorescence anisotropy methods applied to an extrinsic fluorophore that is conjugated to non-native cysteine residues demonstrate that amino acids in an allosteric communication network within a protein subunit tune protein backbone motions at a distal site to enable allosteric binding and inhibition. The unphosphorylated form of the phosphocarrier protein IIAGlc is an allosteric inhibitor of Escherichia coli glycerol kinase, binding more than 25 A from the kinase active site. Crystal structures that showed a ligand-dependent conformational change and large temperature factors for the IIAGlc-binding site on E. coli glycerol kinase suggest that motions of the allosteric site have an important role in the inhibition. Three E. coli glycerol kinase amino acids that are located at least 15 A from the active site and the allosteric site were shown previously to be necessary for transplanting IIAGlc inhibition into the nonallosteric glycerol kinase from Haemophilus influenzae. These three amino acids are termed the coupling locus. The apparent allosteric site motions and the requirement for the distant coupling locus to transplant allosteric inhibition suggest that the coupling locus modulates the motions of the IIAGlc-binding site. To evaluate this possibility, variants of E. coli glycerol kinase and the chimeric, allosteric H. influenzae glycerol kinase were constructed with a non-native cysteine residue replacing one of the native residues in the IIAGlc-binding site. The extrinsic fluorophore Oregon Green 488 (2',7'-difluorofluorescein) was conjugated specifically to the non-native cysteine residue. Steady-state and time-resolved fluorescence anisotropy measurements show that the motions of the fluorophore reflect backbone motions of the IIAGlc-binding site and these motions are modulated by the amino acids at the coupling locus.

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

“…EGK and A65T EGK were purified as described [33, 34]. The plasmid construct for the R369A EGK was generously supplied by Dr. S. James Remington of the University of Oregon.…”

“…The R369A EGK enzyme was purified as described [33]. The site-directed variant EGK enzymes E92C and E121C were constructed as described [34]. These enzymes were purified by using Q-Sepharose HP, Source 15Q, and ATP-agarose (C-8 linkage) affinity chromatographies on an Akta Purifier system.…”

“…The samples were chromatographed on Sephadex G-25 and dialyzed against three changes of standard buffer pH 7 to remove unconjugated fluorophore. After dialysis, the protein concentration and fluorophore label stoichiometry were determined as described [34, 40]. Fluorescence emission spectra at 25°C in 0.1 M triethanolamine HCl buffer at pH 7.0 with 2 mM glycerol were recorded by using an ISS Phoenix spectrofluorometer with the excitation wavelength set to 336 nm.…”

“…Fluorescence emission spectra at 25°C in 0.1 M triethanolamine HCl buffer at pH 7.0 with 2 mM glycerol were recorded by using an ISS Phoenix spectrofluorometer with the excitation wavelength set to 336 nm. To identify the site of conjugation, OG-modified enzyme was reduced, carboxymethylated, and treated with trypsin, and peptides were isolated by using C 18 reverse-phase chromatography as described [34, 40]. Column fractions that contained A 480 -absorbing material were analyzed by the Texas A&M University Protein Chemistry Laboratory by using MALDI-TOF mass spectrometry.…”

Amino acid substitutions in the sugar kinase/hsp70/actin superfamily conserved ATPase core of E. coli glycerol kinase modulate allosteric ligand affinity but do not alter allosteric coupling

“…Approaches for identification of these networks include bioinformatics methods [7, 9, 10], computational methods [2, 4, 11-13], and structure-based experimental methods [5, 14-18]. We used an approach that is based on transplanting allosteric control into a naïve enzyme to identify a locus that is part of an allosteric network [19] and showed that the locus modulates local motions and binding affinity at the allosteric site [20]. Here, we evaluate the role of this locus in the coupling between the allosteric and catalytic sites.…”