Native-state conformational dynamics of GART: a regulatory pH-dependent coil-helix transition examined by electrostatic calculations.
Glycinamide ribonucleotide transformylase (GART) undergoes a pH-dependent coil-helix transition with pK(a) approximately 7. An alpha-helix is formed at high pH spanning 8 residues of a 21-residue-long loop, comprising the segment Thr120-His121-Arg122-Gln123-Ala124-Leu125-Glu126-Asn127. To understand the electrostatic nature of this loop-helix, called the activation loop-helix, which leads to the formation and stability of the alpha-helix, pK(a) values of all ionizable residues of GART have been calculated, using Poisson-Boltzmann electrostatic calculations and crystallographic data. Crystallographic structures of high and low pH E70A GART have been used in our analysis. Low pK(a) values of 5.3, 5.3, 3.9, 1.7, and 4.7 have been calculated for five functionally important histidines, His108, His119, His121, His132, and His137, respectively, using the high pH E70A GART structure. Ten theoretical single and double mutants of the high pH E70A structure have been constructed to identify pairwise interactions of ionizable residues, which have aided in elucidating the multiplicity of electrostatic interactions of the activation loop-helix, and the impact of the activation helix on the catalytic site. Based on our pK(a) calculations and structural data, we propose that: (1) His121 forms a molecular switch for the coil-helix transition of the activation helix, depending on its protonation state; (2) a strong electrostatic interaction between His132 and His121 is observed, which can be of stabilizing or destabilizing nature for the activation helix, depending on the relative orientation and protonation states of the rings of His121 and His132; (3) electrostatic interactions involving His119 and Arg122 play a role in the stability of the activation helix; and (4) the activation helix contains the helix-promoting sequence Arg122-Gln123-Ala124-Leu125-Glu126, but its alignment relative to the N and C termini of the helix is not optimal, and is possibly of a destabilizing nature. Finally, we provide electrostatic evidence that the formation and closure of the activation helix create a hydrophobic environment for catalytic-site residue His108, to facilitate catalysis.
Morikis
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Department of Chemical and Environmental Engineering, University of California at Riverside, Riverside, California 92521-0444, USA. dmorikis@engr.ucr.edu
Elcock
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Jennings
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McCammon
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D000409:Alanine / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D002384:Catalysis; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D019877:Hydroxymethyl and Formyl Transferases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D008433:Mathematics; D008958:Models, Molecular; D009154:Mutation; D051026:Phosphoribosylglycinamide Formyltransferase; D011487:Protein Conformation; D055672:Static Electricity; D017186:Titrimetry
D000596:Amino Acids; D006639:Histidine; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D000409:Alanine