Stereospecific assignments of 1H-nmr methyl lines and conformation of valyl residues in the lac repressor headpiece

Two-dimensional ¹H-nmr methods are described to obtain information on the sidechain conformation of valyl residues of the lac repressor headpiece and to assign the resonances of their methyl groups stereospecifically. The spin–spin coupling constants (J_αβ) between C^αand C^β protons are obtained from two-dimensional correlated spectroscopy experiments. Large values for J_αβ(10–12 Hz) corresponding to trans orientations for these protons (g⁺ conformation) are found for all valyl residues in α-helical segments. For these valyl residues, the distance between one methyl group (γ₁)and the valyl amide proton is much shorter than for the other methyl group, so that stereospecific resonance assignments follow from relative intensities of the corresponding cross peaks in a two-dimensional nuclear Overhauser enhancement spectrum. Thus, streospecific assignments could be made for the methyl groups of Val 9, 20, 23, and 38 (of a total of eight valyl residues).     

Determination of protein structures from nuclear magnetic resonance data using a restrained molecular dynamics approach: the lac repressor DNA binding domain

A procedure is described to determine from NMR data the three-dimensional structure of biomolecules. This procedure combines model building with a restrained Molecular Dynamics algorithm, in which distance information from NOEs is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or "headpiece" (amino acids 1-51) of the lac repressor from E. coli, for which no crystal structure is available. The spatial structure of the headpiece is discussed in terms of known physical and biochemical data and of its DNA binding properties.     

Spatial arrangement of the three alpha helices in the solution conformation of E. coli lac repressor DNA-binding domain

The relative orientations of the 3 helices in the DNA-binding domain ('headpiece') of lac repressor have been determined using distance constraints obtained from 2-dimensional 1H nuclear Overhauser enhancement spectra. The relative orientations of its helices is similar to that of the central 3 helices in the DNA-binding domain of the lambda repressor of the bacteriophage lambda.     

The binding of protons and inositol hexakisphosphate to ligated and unligated human des-Arg141alpha-hemoglobin.

The region of Bacillus stearothermophilus strain NCA 1503 23-S ribosomal RNA protected from T1 ribonuclease digestion by the 50-S ribosomal subunit protein L1 from Escherichia coli has been established. The sequence of 115 nucleotides is compared to the analogous region in E. coli. The similar behaviour of the RNA towards the recognition of protein L1 may be explained in terms of secondary base-pairing, even though there exists almost 40% difference between the primary nucleotide sequences.     

Mapping the binding interface of the cytochrome b5-cytochrome c complex by nuclear magnetic resonance

The interaction between bovine cytochrome b(5) (cyt b(5)) and horse heart cytochrome c (cyt c) is investigated by NMR spectroscopy. Chemical shifts of cyt b(5) backbone resonances and side chain methyl resonances were monitored as a function of cyt c concentration. The shifts are small but saturatable and indicate that the binding of cyt b(5) with cyt c is in fast exchange. An equilibrium association constant of (6 +/- 3) x 10(4) M(-1) was obtained with a lower limit of 180 s(-1) for the dissociation rate of the complex. To resolve considerable ambiguities in the interpretation of the chemical shift mapping, (15)N relaxation experiments and cross-saturation experiments were used as alternative methods to map the cyt b(5)-cyt c binding interface. Results from the three experiments combined demonstrate that the conserved negatively charged region of cyt b(5) surrounding the solvent-exposed heme edge is involved in the interaction with cyt c. These data support the models proposed by Salemme and Mauk [(1976) J. Mol. Biol. 102, 563-568; (1993) Biochemistry 32, 6613-6623].     

Backbone dynamics of the ribonuclease binase active site area using multinuclear ((15)N and (13)CO) NMR relaxation and computational molecular dynamics

The nano-pico second backbone dynamics of the ribonuclease binase, homologous to barnase, is investigated with (15)N, (13)C NMR relaxation at 11.74 and 18.78 T and with a 1.1 ns molecular dynamics simulation. The data are compared with the temperature factors reported for the X-ray structure of this enzyme. The molecular dynamics and X-ray data correspond well and predict motions in the loops 56-61 and 99-104 that contain residues that specifically recognize substrate and are catalytic (His101), respectively. In contrast, the (15)N relaxation data indicate that these loops are mostly ordered at the nano-pico second time scale. Nano-pico second motions in the recognition loop 56-61 are evident from (13)CO-(13)C cross relaxation data, but the mobility of the catalytic loop 99-104 is not detected by (13)CO cross relaxation either. From the results of this and previous work [Wang, L., Pang, Y., Holder, T., Brender, J. R., Kurochkin, A., and Zuiderweg, E. R. P. (2001) Proc. Natl. Acad. Sci. U.S.A., 98, 7684-7689], the following dynamical characterization of the active site area of binase emerges: a beta sheet, rigid at all probed time scales, supports the catalytic residue Glu 72. Both substrate-encapsulating loops are mobile on both fast and slow time scales, but the fast motions of the loop which contains the other catalytic residue, His 101, as predicted by B-factors and computational molecular dynamics is not detected by NMR relaxation. This work strongly argues for the use of several measures in the study of protein dynamics.     

Solution structure and phosphopeptide binding to the N-terminal domain of Yersinia YopH: comparison with a crystal structure

Virulence of pathogenic bacteria of the genus Yersinia requires the injection of six effector proteins into the cytoplasm of host cells. The amino-terminal domain of one of these effectors, the tyrosine phosphatase YopH, is essential for translocation of YopH, as well as for targeting it to phosphotyrosine-containing substrates of the type pYxxP. We report the high-resolution solution structure of the N-terminal domain (residues 1-129) from the Yersinia pseudotuberculosis YopH (YopH-NT) in complex with N-acetyl-DEpYDDPF-NH(2), a peptide derived from an in vivo protein substrate. In contrast to the domain-swapped dimer observed in a crystal structure of the same protein (Smith, C. L., Khandelwal, P., Keliikuli, K., Zuiderweg, E. R. P., and Saper, M. A. (2001) Mol. Microbiol. 42, 967-979), YopH-NT is monomeric in solution. The peptide binding site is located on a beta-hairpin that becomes the crossover point in the dimer structure. The binding site has several characteristics that are reminiscent of SH2 domains, which also bind to pYxxP sequences.     

Structural insights into substrate binding by the molecular chaperone DnaK

How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones. We find here that the Escherichia coli Hsp70, DnaK, lacking the entire alpha-helical domain, DnaK(1-507), retains the ability to support lambda phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro. We determined the NMR solution structure of the corresponding substrate binding domain, DnaK(393-507), without substrate, and assessed the impact of substrate binding. Without bound substrate, loop L3,4 and strand beta3 are in significantly different conformations than observed in previous structures of the bound DnaK substrate binding domain, leading to occlusion of the substrate binding site. Upon substrate binding, the beta-domain shifts towards the structure seen in earlier X-ray and NMR structures. Taken together, our results suggest that conformational changes in the beta-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity.