NMR analysis of [methyl-13C]methionine UvrB from Bacillus caldotenax reveals UvrB-domain 4 heterodimer formation in solution.
UvrB is a central DNA damage recognition protein involved in bacterial nucleotide excision repair. Structural information has been limited by the apparent disorder of the C-terminal domain 4 in crystal structures of intact UvrB; in solution, the isolated domain 4 is found to form a helix-loop-helix dimer. In order to gain insight into the behavior of UvrB in solution, we have performed NMR studies on [methyl-13C]methionine-labeled UvrB from Bacillus caldotenax (molecular mass=75 kDa). The 13 methyl resonances were assigned on the basis of site-directed mutagenesis and domain deletion. Solvent accessibility was assessed based on the relaxation and chemical shift responses of the probe methyl resonances to the stable nitroxide, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL). M632, located at the potential dimer interface of domain 4, provides an ideal probe for UvrB dimerization behavior. The M632 resonance of UvrB is very broad, consistent with some degree of monomer-dimer exchange and/or conformational instability of the exposed dimer interface. Upon addition of unlabeled domain 4 peptide, the M632 resonance of UvrB sharpens and shifts to a position consistent with a UvrB-domain 4 heterodimer. A dissociation constant (KD) value of 3.3 microM for the binding constant of UvrB with the domain 4 peptide was derived from surface plasmon resonance studies. Due to the flexibility of the domain 3-4 linker, inferred from limited proteolysis data and from the relaxation behavior of linker residue M607, the position of domain 4 is constrained not by the stiffness of the linking segment but by direct interactions with domains 1-3 in UvrB. In summary, UvrB homodimerization is disfavored, while domain 4 homodimerization and UvrB-domain 4 heterodimerization are allowed.
DellaVecchia
Matthew J
MJ
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA.
Merritt
W Keither
WK
Peng
Ye
Y
Kirby
Thomas W
TW
DeRose
Eugene F
EF
Mueller
Geoffrey A
GA
Van Houten
Bennett
B
London
Robert E
RE
D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002247:Carbon Isotopes; D045643:DNA Repair Enzymes / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D012996:Solutions; D020349:Surface Plasmon Resonance