Molecular basis for complex formation between methylamine dehydrogenase and amicyanin revealed by inverse mutagenesis of an interprotein salt bridge.
Methylamine dehydrogenase (MADH) and amicyanin form a physiologic complex which is required for interprotein electron transfer. The crystal structure of this protein complex is known, and the importance of certain residues on amicyanin in its interaction with MADH has been demonstrated by site-directed mutagenesis. In this study, site-directed mutagenesis of MADH, kinetic data, and thermodynamic analysis are used to probe the molecular basis for stabilization of the protein complex by an interprotein salt bridge between Arg99 of amicyanin and Asp180 of the alpha subunit of MADH. This paper reports the first site-directed mutagenesis of MADH, as well as the construction, heterologous expression, and characterization of a six-His-tagged MADH. alpha Asp180 of MADH was converted to arginine to examine the effect on complex formation with native and mutant amicyanins. This mutation had no effect on the parameters for methylamine oxidation by MADH, but significantly affected its interaction with amicyanin. Of the native and mutant proteins that were studied, their observed order of affinity for each other was as follows: native MADH and native amicyanin > native MADH and R99D amicyanin > alpha D180R MADH and native amicyanin > alpha D180R MADH and R99D amicyanin, and alpha D180R MADH and R99L amicyanin. The alpha D180R mutation also eliminated the ionic strength dependence of the reaction of MADH with amicyanin that is observed with wild-type MADH. Interestingly, the inverse mutation pair of alpha D180R MADH and R99D amicyanin did not restore the favorable salt bridge, but instead disrupted complex formation much more severely than did either individual mutation. These results are explained using molecular modeling and thermodynamic analysis of the kinetic data to correlate the energy contributions of specific stabilizing and destabilizing interactions that are present in the wild-type and mutant complexes. A model is also proposed to describe the sequence of events that leads to stable complex formation between MADH and amicyanin.
Department of Biochemistry, The University of Mississippi Medical Center, Jackson, Mississippi 39216-4505, USA.
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Z
Zhu
L H
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Jones
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Graichen
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Davidson
D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; C017079:mauC protein, Methylobacterium extorquens; D001224:Aspartic Acid; D006639:Histidine; D001120:Arginine; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors