Crystal structure, folding, and operator binding of the hyperstable Arc repressor mutant PL8.
Arc repressor is a small, dimeric DNA-binding protein that belongs to the ribbon-helix-helix family of transcription factors. Replacing Pro8 at the N-terminal end of the beta-sheet with leucine increases the stability of the mutant protein by 2.5 kcal/mol of dimer. However, this enhanced stability is achieved at the expense of significantly reduced DNA binding affinity. The structure of the PL8 mutant dimer has been determined to 2.4-A resolution by X-ray crystallography. The overall structure of the mutant is very similar to wild type, but Leu8 makes an additional interstrand hydrogen bond at each end of the beta-sheet of the mutant, increasing the total number of beta-sheet hydrogen bonds from six to eight. Comparison of the refolding and unfolding kinetics of the PL8 mutant and wild-type Arc shows that the enhanced stability of the mutant is accounted for by a decrease in the rate of protein unfolding, suggesting that the mutation acts to stabilize the native state and that the beta-sheet forms after the rate-limiting step in folding. The reduced operator affinity of the PL8 dimer appears to arise because the mutant cannot make the new interstrand hydrogen bonds and simultaneously make the wild-type set of contacts with operator DNA.
Schildbach
J F
JF
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Milla
M E
ME
Jeffrey
P D
PD
Raumann
B E
BE
Sauer
R T
RT
D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000648:ultrastructure; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D006358:Hot Temperature; D006860:Hydrogen Bonding; D009875:Operator Regions, Genetic; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000378:metabolism* / Q000648:ultrastructure; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014764:Viral Proteins / Q000378:metabolism* / Q000648:ultrastructure; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D006146:Guanidines; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D019791:Guanidine