Michael J. Osborne

Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes

Heteronuclear NMR methods have been used to probe the conformation of four complexes of Escherichia coli dihydrofolate reductase (DHFR) in solution. 1HN, 15N, and 13Cα resonance assignments have been made for the ternary complex with folate and oxidized NADP+ cofactor and the ternary complex with folate and a reduced cofactor analog, 5,6‐dihydroNADPH. The backbone chemical shifts have been compared with those of the binary complex of DHFR with the substrate analog folate and the binary complex with NADPH (the holoenzyme). Analysis of 1HN and 15N chemical shifts has led to the identification of marker resonances that report on the active site conformation of the enzyme. Other backbone amide resonances report on the presence of ligands in the pterin binding pocket and in the adenosine and nicotinamide–ribose binding sites of the NADPH cofactor. The chemical shift data indicate that the enzyme populates two dominant structural states in solution, with the active site loops in either the closed or occluded conformations defined by X‐ray crystallography; there is no evidence that the open conformation observed in some X‐ray structures of E. coli DHFR are populated in solution.

Structural dynamics of the membrane translocation domain of colicin E9 and its interaction with TolB

In order for the 61 kDa colicin E9 protein toxin to enter the cytoplasm of susceptible cells and kill them by hydrolysing their DNA, the colicin must interact with the outer membrane BtuB receptor and Tol translocation pathway of target cells. The translocation function is located in the N-terminal domain of the colicin molecule. (1)H, (1)H-(1)H-(15)N and (1)H-(13)C-(15)N NMR studies of intact colicin E9, its DNase domain, minimal receptor-binding domain and two N-terminal constructs containing the translocation domain showed that the region of the translocation domain that governs the interaction of colicin E9 with TolB is largely unstructured and highly flexible. Of the expected 80 backbone NH resonances of the first 83 residues of intact colicin E9, 61 were identified, with 43 of them being assigned specifically. The absence of secondary structure for these was shown through chemical shift analyses and the lack of long-range NOEs in (1)H-(1)H-(15)N NOESY spectra (tau(m)=200 ms). The enhanced flexibility of the region of the translocation domain containing the TolB box compared to the overall tumbling rate of the protein was identified from the relatively large values of backbone and tryptophan indole (15)N spin-spin relaxation times, and from the negative (1)H-(15)N NOEs of the backbone NH resonances. Variable flexibility of the N-terminal region was revealed by the (15)N T(1)/T(2) ratios, which showed that the C-terminal end of the TolB box and the region immediately following it was motionally constrained compared to other parts of the N terminus. This, together with the observation of inter-residue NOEs involving Ile54, indicated that there was some structural ordering, resulting most probably from the interactions of side-chains. Conformational heterogeneity of parts of the translocation domain was evident from a multiplicity of signals for some of the residues. Im9 binding to colicin E9 had no effect on the chemical shifts or other NMR characteristics of the region of colicin E9 containing the TolB recognition sequence, though the interaction of TolB with intact colicin E9 bound to Im9 did affect resonances from this region. The flexibility of the translocation domain of colicin E9 may be connected with its need to recognise protein partners that assist it in crossing the outer membrane and in the translocation event itself.

Association between the first two immunoglobulin-like domains of the neural cell adhesion molecule N-CAM

The extracellular domain of N‐CAM contains five immunoglobulin‐like (Ig) and two fibronectin type III‐like domains and facilitates cell‐cell binding through multiple, weak interdomain interactions. NMR spectroscopy indicated that the two N‐terminal Ig‐like domains from chicken N‐CAM (Ig I and Ig II) interact with millimolar affinity. Physico‐chemical studies show that this interaction is significantly amplified when the domains are covalently linked, consistent with an antiparallel domain arrangement. The binding of the two individual domains and the dimerization of the concatenated protein were essentially independent of salt, up to a concentration of 200 mM. The residues in Ig I involved in the interaction map to the BED strands of the β sandwich, and delineate a largely hydrophobic patch.

Anisotropic rotational diffusion in model-free analysis for a ternary DHFR complex.

Model-free analysis has been extensively used to extract information on motions in proteins over a wide range of timescales from NMR relaxation data. We present a detailed analysis of the effects of rotational anisotropy on the model-free analysis of a ternary complex for dihydrofolate reductase (DHFR). Our findings show that the small degree of anisotropy exhibited by DHFR (D||/D⊥=1.18) introduces erroneous motional models, mostly exchange terms, to over 50% of the NH spins analyzed when isotropic tumbling is assumed. Moreover, there is a systematic change in S2, as large as 0.08 for some residues. The significant effects of anisotropic rotational diffusion on model-free motional parameters are in marked contrast to previous studies and are accentuated by lowering of the effective correlation time using isotropic tumbling methods. This is caused by the preponderance of NH vectors aligned perpendicular to the principal diffusion tensor axis and is readily detected because of the high quality of the relaxation data. A novel procedure, COPED (COmparison of Predicted and Experimental Diffusion tensors) is presented for distinguishing genuine motions from the effects of anisotropy by comparing experimental relaxation data and data predicted from hydrodynamic analyses. The procedure shows excellent agreement with the slow motions detected from the axially symmetric model-free analysis and represents an independent procedure for determining rotational diffusion and slow motions that can confirm or refute established procedures that rely on relaxation data. Our findings show that neglect of even small degrees of rotational diffusion anisotropy can introduce significant errors in model-free analysis when the data is of high quality. These errors can hinder our understanding of the role of internal motions in protein function.

Determination of the [Fe4S4]Cys4 cluster geometry of Desulfovibrio africanus ferredoxin I by 1H NMR spectroscopy

1D and 2D 1H NMR studies of the Fe4S4, cluster containing ferredoxin I from Desulfovibrio africanus have been carried out with the aim of determining the geometry of the cluster linkages with the 4 Cys side chains that bind the cluster. This required the Cys βCH resonances of the oxidised protein to be sequence‐specifically and stereo‐specifically assigned, and this was accomplished by a combination of TOCSY and NOE measurements, allied to model building based on X‐ray structures of related ferredoxins. An analysis of the estimated hyperfine shifts of the Cys βCH resonances with a Karplus‐type equation relating the shifts to iron‐sulfur‐β carbon‐/3 proton dihedral angles, taken together with the relative relaxation rates of the two βCH2 resonances, estimated from their linewidths, then allowed the ironsulfur‐/3‐carbon‐α‐carbon dihedral angles to be determined. A novel representation of the NMR data is presented which shows that the cluster dihedral angles are uniquely determined by the NMR data. The analysis reveals that the dihedral angles for D. africanus ferredoxin I are similar to the corresponding angles of other ferrredoxins even though there are differences in their 1H NMR spectra. The sequence‐specific and stereospecific assignments have been extended by analogy to the related Fe4S4‐containing D. gigas ferredoxin I, and the stereospecific assignments to the Fe4S4‐containing Thermococcus litoralis ferredoxin.

Solution Structure of YaeO, a Rho-specific Inhibitor of Transcription Termination

Rho-dependent transcription termination is an essential process for the regulation of bacterial gene expression. Thus far, only two Rho-specific inhibitors of bacterial transcription termination have been described, the psu protein from the satellite bacteriophage P4 and YaeO from Escherichia coli. Here, we report the solution structure of YaeO, the first of a Rho-specific inhibitor of transcription termination. YaeO is an acidic protein composed of an N-terminal helix and a seven-stranded β sandwich. NMR chemical shift perturbation experiments revealed that YaeO binds proximal to the primary nucleic acid binding site of Rho. Based on the NMR titrations, a docked model of the YaeO-Rho complex was calculated. These results suggest that YaeO binds outside the Rho hexamer, acting as a competitive inhibitor of RNA binding. In vitro gel shift assays confirmed the inhibition of nucleic acid binding to Rho. Site-directed mutagenesis showed that the negative character of YaeO is essential for its function in vivo.

The solution structure of YbcJ from Escherichia coli reveals a recently discovered alphaL motif involved in RNA binding.

The structure of the recombinant Escherichia coli protein YbcJ, a representative of a conserved family of bacterial proteins (COG2501), was determined by nuclear magnetic resonance. The fold of YbcJ identified it as a member of the larger family of S4-like RNA binding domains. These domains bind to structured RNA, such as that found in tRNA, rRNA, and a pseudoknot of mRNA. The structure of YbcJ revealed a highly conserved patch of basic residues, comprising amino acids K26, K38, R55, K56, and K59, which likely participate in RNA binding.

MCD and 1H-NMR spectroscopic studies of Desulfovibrio africanus ferredoxin I: revised amino-acid sequence and identification of secondary structure

Desulfovibrio africanus ferredoxin I was studied by magnetic circular dichroism and 1H-NMR spectroscopies. These showed the presence of histidine and tryptophan, in contrast to the previously reported amino-acid sequence (Bruschi and Hatchikian (1982) Biochimie 64, 503-507). This was redetermined and the revised sequence shown to contain both histidine and tryptophan, as well as four other corrections (Sery et al. (1994) Biochemistry, submitted). Electrospray mass spectrometry confirmed the mass of the ferredoxin was that given by the revised amino-acid sequence. The secondary structure of the ferredoxin I was investigated with two-dimensional 1H-NMR experiments and both alpha-helix and beta-sheet structure detected. The influence of the paramagnetism of the Fe4 S4 cluster on the NMR properties of the ferredoxin protons was investigated, by temperature-dependent experiments, and it was concluded that there is only a negligible dipolar contribution to resonance chemical shifts from this source. The significance of this for the determination of the three-dimensional structure of the ferredoxin by NMR is discussed.

NMR Studies of Paramagnetic Systems to Characterise Small Molecule:Protein and Protein:Protein Interactions

Intermolecular interactions are a feature of all biochemical processes, and whilst some involve the interaction of small molecules with biopolymers or membranes, others involve the interaction of two or more biopolymers. In all cases, a thorough description of the molecular details of the interaction requires kinetic, thermodynamic and structural investigations. In general, however, there are a limited number of possible kinetic and thermodynamic schemes.

A 1H-NMR study of the interactions between rat tissue kallikrein and two peptide inhibitors

The 1H-NMR spectra have been obtained for rat submandibular kallikrein in the absence and presence of inhibitors. Two competitive inhibitors were investigated, the tripeptide leupeptin (a potent inhibitor with Ki 0.5 microM) and a hexapeptide (a much weaker, substrate-analogue inhibitor with Ki 380 microM). Analysis of the NMR spectra showed that binding of leupeptin to kallikrein led to a change in the conformation of the enzyme, whereas binding of the substrate analogue to the enzyme produced no such change and may have resulted in a conformational change of the inhibitor.