Structure and orientation of T4 lysozyme bound to the small heat shock protein alpha-crystallin.
We have determined the structural changes that accompany the formation of a stable complex between a destabilized mutant of T4 lysozyme (T4L) and the small heat shock protein alpha-crystallin. Using pairs of fluorescence or spin label probes to fingerprint the T4L tertiary fold, we demonstrate that binding disrupts tertiary packing in the two domains as well as across the active-site cleft. Furthermore, increased distances between i and i+4 residues of helices support a model in which the bound structure is not native-like but significantly unfolded. In the confines of the oligomer, T4L has a preferential orientation with residues in the more hydrophobic C-terminal domain sequestered in a buried environment, while residues in the N-terminal domain are exposed to the aqueous solvent. Furthermore, electron paramagnetic resonance spectral line shapes of sites in the N-terminal domain are narrower than in the folded, unbound T4L reflecting an unstructured backbone and an asymmetric pattern of contacts between T4L and alpha-crystallin. The net orientation is not affected by the location of the destabilizing mutation consistent with the notion that binding is not triggered by recognition of localized unfolding. Together, the structural and thermodynamic data indicate that the stably bound conformation of T4L is unfolded and support a model in which the two modes of substrate binding originate from two discrete binding sites on the chaperone.
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 741 Light Hall, 2215 Garland Avenue, Nashville, TN 37232, USA.
Derek P
DP
Claxton
Ping
P
Zou
Hassane S
HS
Mchaourab
D017122:Bacteriophage T4 / Q000201:enzymology*; D001665:Binding Sites; D001643:Bridged Bicyclo Compounds / Q000737:chemistry; D003001:Cloning, Molecular; D003497:Cyclic N-Oxides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D050888:Heat-Shock Proteins, Small / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008954:Models, Biological; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D013379:Substrate Specificity; D013816:Thermodynamics; D014867:Water / Q000737:chemistry; D038201:alpha-Crystallins / Q000302:isolation & purification / Q000378:metabolism*
D001643:Bridged Bicyclo Compounds; D003497:Cyclic N-Oxides; D005456:Fluorescent Dyes; D050888:Heat-Shock Proteins, Small; D012997:Solvents; D013113:Spin Labels; D038201:alpha-Crystallins; D014867:Water; C029910:3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl; D009113:Muramidase; C028381:monobromobimane