A model for the solution structure of oxidized putidaredoxin (Pdx), a 106-residue globular protein containing a Fe2S2 cluster, has been determined using homonuclear NMR methods. Pdx is the first of the class of Fe2S2Cys4 ferredoxins which act as electron-transfer partners for P-450 monooxygenases to be structurally characterized, and no crystal structure has been determined for Pdx or for any closely homologous protein. Pdx is the physiological redox partner of cytochrome P-450cam. A total of 878 NOE distance constraints, 66 phi angular constraints derived from NH-C alpha H coupling constants, and five paramagnetic broadening constraints were used in simulated annealing structural refinements to obtain a family of structures with pairwise rms deviations of 1.14 A for backbone atoms and 1.80 A for all non-hydrogen atoms. Paramagnetic broadening of resonances within a ca. 8-A radius of the metal cluster prevents the use of NMR-derived constraints in this region of the protein; structural constraints used to model the environment of the metal cluster were obtained from site-directed mutagenesis and model compounds and by comparison with known ferredoxin structures. Pdx retains a similar folding topology to other structurally characterized Fe2S2Cys4 ferredoxins but differs from the other ferredoxins in containing a significantly more compact structure in the C-terminal half of the protein.
The WW domain adopts a compact, three-stranded, antiparallel beta-sheet structure that mediates protein-protein interactions by binding to xPPxY-based protein ligands, such as the PY-ligand (EYPPYPPPPYPSG) derived from p53 binding protein-2. The conserved Trp residues, after which this domain was named, were replaced with Phe so their importance in structural integrity and for ligand binding could be evaluated. A biophysical approach was employed to compare the W17F, W39F, and W17F/W39F WW domains to the wild-type protein. The data demonstrate that replacement of Trp39 with Phe (W39F) does not disrupt the structure of the WW domain variant, but does abolish ligand binding. In contrast, the W17F WW domain variant is largely if not completely unfolded; however, this variant undergoes a PY-ligand induced disorder to order (folding) transition. The dissociation constant for the W17F WW domain-PY-ligand interaction is 15.1 +/- 1.2 microM, only slightly higher than that observed for the wild-type WW domain interaction (5.9 +/- 0.33 microM). The W17F WW domain is a natively unfolded protein which adopts a native conformation upon PY-ligand binding.
Multidimensional NMR methods were used to obtain 'H-I'N correlations and "N resonance assignments for amide and side-chain nitrogens of oxidized and reduced putidaredoxin (Pdx), the Fe2S2 ferredoxin, which acts as the physiological reductant of cytochrome P-450,,, (CYPlOl). A model for the solution structure of oxidized Pdx has been determined recently using NMR methods (Pochapsky TC, Ye XM, Ratnaswamy G, Lyons TA, 1994, Biochemistry 33:6424-6432) and redox-dependent ' H NMR spectral features have been described (Pochapsky TC, Ratnaswamy G, Patera A, 1994, Biochemistry33:6433-6441). "N assignments were made with NOESY-('H/''N) HMQC and TOCSY-('H/''N) HSQC spectra obtained using samples of Pdx uniformly labeled with "N. Local dynamics in both oxidation states of Pdx were then characterized by comparison of residue-specific amide proton exchange rates, which were measured by a combination of saturation transfer and H 2 0 / D 2 0 exchange methods at pH 6.4 and 7.4 (uncorrected for isotope effects). In general, where exchange rates for a given site exhibit significant oxidation-state dependence, the oxidized protein exchanges more rapidly than the reduced protein. The largest dependence of exchange rate upon oxidation state is found for residues near the metal center and in a region of compact structure that includes the loop-turn Val 74-Ser 82 and the C-terminal residues (Pro 102-Trp 106). The significance of these findings is discussed in light of the considerable dependence of the binding interaction between Pdx and CYPlOl upon the oxidation state of Pdx.
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