Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy.
The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694], appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase [Sachsenheimer, W., & Schulz, G.E. (1977) J. Mol. Biol. 114, 23-26], with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of beta-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% alpha-helix, 38% beta-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possibly due to disorder, it can be fit by using methods developed on well-characterized globular proteins. On this basis, the peptide consists of 35 +/- 10% beta-structure, 60 +/- 12% turns and aperiodic structure, and not more than 10% alpha-helix. The CD spectrum is best fit by assuming the presence of at most 13% alpha-helix in the peptide, 24 +/- 2% beta-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformational changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assessed by CD. Detailed assignments of resonances in the peptide spectrum and intermolecular NOEs between protons of bound MgATP and those of the peptide, as well as chemical shifts of peptide resonances induced by the binding of MgATP, are consistent with the previously proposed binding site for MgATP on adenylate kinase.
Fry
D C
DC
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Byler
D M
DM
Susi
H
H
Brown
E M
EM
Kuby
S A
SA
Mildvan
A S
AS
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000263:Adenylate Kinase / Q000378:metabolism*; D000818:Animals; D002942:Circular Dichroism; D005583:Fourier Analysis; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D012996:Solutions; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet
D010446:Peptide Fragments; D012996:Solutions; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D000263:Adenylate Kinase