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Native-state conformational dynamics of GART: A regulatory pH-dependent coil–helix transition examined by electrostatic calculations

¹ Department of Chemical and Environmental Engineering, University of California at Riverside, Riverside, California 92521-0444, USA
² Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242-1109, USA
³ Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0365, USA
⁴ Department of Pharmacology and Howard Hughes Medical Institute, University of California at San Diego, La Jolla, California 92093-0365, USA

Reprint requests to: Dr. Dimitrios Morikis, Department of Chemical and Environmental Engineering, University of California at Riverside, Riverside, CA 92521-0444, USA; e-mail: dmorikis{at}engr.ucr.edu; fax: (909) 787-2425.

Glycinamide ribonucleotide transformylase (GART) undergoes a pH-dependent coil–helix transition with pK_a 7. An -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 -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 idey 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.

Keywords: GART ; glycinamide ribonucleotide transformylase ; electrostatic calculations ; Poisson-Boltz-mann ; pKa ; helix-coil transition

Abbreviations: GAR, glycinamide ribonucleotide • GART, GAR transformulase • PDB, Protein Data Bank

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