Structural and thermodynamic characterization of the DNA binding properties of a triple alanine mutant of MATalpha2.
Triply mutated MATalpha2 protein, alpha2-3A, in which all three major groove-contacting residues are mutated to alanine, is defective in binding DNA alone or in complex with Mcm1 yet binds with MATa1 with near wild-type affinity and specificity. To gain insight into this unexpected behavior, we determined the crystal structure of the a1/alpha2-3A/DNA complex. The structure shows that the triple mutation causes a collapse of the alpha2-3A/DNA interface that results in a reorganized set of alpha2-3A/DNA contacts, thereby enabling the mutant protein to recognize the wild-type DNA sequence. Isothermal titration calorimetry measurements reveal that a much more favorable entropic component stabilizes the a1/alpha2-3A/DNA complex than the alpha2-3A/DNA complex. The combined structural and thermodynamic studies provide an explanation of how partner proteins influence the sequence specificity of a DNA binding protein.
Ke
Ailong
A
Department of Biophysics and Biophysical Chemistry, John Hopkins University, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Mathias
Jonathan R
JR
Vershon
Andrew K
AK
Wolberger
Cynthia
C
D000409:Alanine / Q000737:chemistry*; D002151:Calorimetry; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry*; D018398:Homeodomain Proteins / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry*; D013816:Thermodynamics
D018398:Homeodomain Proteins; D012097:Repressor Proteins; D004247:DNA; D000409:Alanine