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Brownian dynamics of rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mutants binding rabbit F-actin.

Introduction

F-actin, the polymerized form of actin, can play a significant role in cellular metabolism by enhancing specificity of reactions in various metabolic pathways. F-actin has been shown to bind glycolytic enzymes like fructose-l,6- biphosphate aldolase (aldolase) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). These interactions between F-actin and glycolytic enzymes have been suggested to be electrostatic in nature (1) Previous studies involving GAPDH and F-actin (2) identified four residues on the GAPDH structure to be important for the binding of the enzyme to F-actin. These residues, lysines 24, 69, 110 and 114 are positively charged and present the source of strong attraction for negative sites on F-actin. Herein, the interactions between computational mutants of GAPDH (one or more of these lysines are replaced with alanine) and F-actin are examined by Brownian dynamics.

Methods

Brownian Dynamics (BD) has been used to study the binding of GAPDH mutants to F-actin. The model of F-actin was built by the method of Holmes et al (3). The tertiary structure of rabbit GAPDH was built by homology modeling using Insight II (Accelyrs, San Diego, CA). Mutants of GAPDH were made using the MacroDox package (4) by replacing charged residues with alanine. The MacroDox Charge set was used to calculate and assign charges to titratable amino acids and determine the electrostatic fields around the proteins. BD simulations determined the potential of mean force and average electrostatic potential of GAPDH mutants in the F-actin electric field as function of the reaction coordinate [R.sub.c] (distance of GAPDH center of mass from the F-actin axis). Calculations were done at pH = 7.0, ionic strength I = 0.05 M, temperature = 298 K.

Results

Table 1 shows the charges and interaction free energies for wild and mutant GAPDHs. The total charge of wild GAPDH was +17.8 e. Single mutations of the four lysine residues on GAPDH reduced the net charge by about 4 e. This resulted in a decrease in binding; i.e., free energy increased from -0.81 to about-0.4 kcal/mol for the single mutants. Net charge and binding decreased even more for the double mutants. No binding was noticed with the triple and quadruple mutants, indicating that by the time the GAPDH charge reaches +7 e binding is eliminated. These results confirm that the interactions of F-actin with GAPDH are indeed electrostatic. The decrease in binding with decrease in charge was, however, not linear indicating that charge alone is not sufficient to explain the binding. The most exposed residues are most important for binding; the most important residue for binding was lysine 69.

Support for this project was provided by NIH/NIGMS 2R 15 GM55929-02.

(1.) Knull HR., Walsh J. (1992) Curr Top Cell Regul, 33, 15-30.

(2.) Ouporov IV., Knull HR., Lowe SL., Thomasson KA. (2001) J Mol Recognit, 14, 29-41.

(3.) Holmes KC., Popp D., Gebhard W., Kabsch W. (1990) Nature, 347, 44-49.

(4.) Northrup SH., Laughner T., Stevenson G. (1997) MacroDox Macromolecular Simulation Program. Tennessee Technological University, Department of Chemistry, Cookeville, TN 38505.

Victor F. Waingeh *, Stephen L. Lowe, Kathryn A. Thomasson

Department of Chemistry, University of North Dakota, Grand Forks, ND 58202
Table 1: Calculated Charges and Free Energies of GAPDH mutants Binding
F-actin. [R.sub.Cmin] is the location along the reaction coordinate
where the minimum in the free energy occurs; i.e., where the
GAPDH/actin interaction is greatest. Only the weakest and strongest
binding results are shown.

 Binding
 Free Energy [R.sub.Cmin]
GAPDH Charge (e) (kcal/mol) ([Angstrom])

Wild-type 17.8 -0.81 82
Single mutants K69A 14.2 -0.35 87
 K110A 13.7 -0.49 81
Double mutants K110_114A 10.0 -0.28 80
 K114_69A 10.4 -0.07 86
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Title Annotation:COMMUNICATIONS: GRADUATE
Author:Waingeh, Victor F.; Lowe, Stephen L.; Thomasson, Kathryn A.
Publication:Proceedings of the North Dakota Academy of Science
Article Type:Report
Geographic Code:1USA
Date:Apr 1, 2002
Words:617
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