Biomedical subjects
Publications and source records attributed to G Otting.
NK-lysin is the first representative of a family of sequence related proteins--saposins, surfactant-associated protein B, pore forming amoeba proteins, and domains of acid sphingomyelinase, acyloxyacylhydrolase and plant aspartic proteinases--for which a structure has been determined.
The structure of the monomeric DNA-binding domain of the Escherichia coli arginine repressor, ArgR, determined by NMR spectroscopy, shows structural homology to the winged helix-turn-helix (wHTH) family, a motif found in a diverse class of proteins including both gene regulators and gene organizers from prokaryotes and eukaryotes. Biochemical data on DNA binding by intact ArgR are used as constraints to position the domain on its DNA target and to derive a model for the hexamer-DNA complex using the known structure of the L-arginine-binding domain. The structural independence of the wHTH fold may be important for multimeric DNA-binding proteins that contact extended DNA regions with imperfect match to consensus sequences, a feature of many wHTH-domain proteins.
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The three-dimensional solution structure of the 1:1 complex between the synthetic peptide ZF-1 and zinc was determined by 1H NMR spectroscopy. The peptide, initially isolated from pig intestines, is identical in sequence to the 30 N-terminal amino acid residues of the human protein Lasp-1 belonging to the LIM domain protein family. The final set of 20 energy-refined NMR conformers has an average rmsd relative to the mean structure of 0.55 A for the backbone atoms of residues 3-30. Calculations without zinc atom constraints unambiguously identified Cys 5, Cys 8, His 26, and Cys 29 as the zinc-coordinating residues. LIM domains consist of two sequential zinc-binding modules and the NMR structure of the ZF-1-zinc complex is the first example of a structure of an isolated module. Comparison with the known structures of the N-terminal zinc-binding modules of both the second LIM domain of chicken CRP and rat CRIP with which ZF-1 shares 50% and 43% sequence identity, respectively, supports the notion that the zinc-binding modules of the LIM domain have a conserved structural motif and identifies local regions of structural diversity. The similarities include conserved zinc-coordinating residues, a rubredoxin knuckle involving Cys 5 and Cys 8, and the coordination of the zinc ion by histidine N delta in contrast to the more usual coordination by N epsilon observed for other zinc-finger domains. The present structure determination of the ZF-1-zinc complex establishes the N-terminal half of a LIM domain as an independent folding unit. The structural similarities of N- and C-terminal zinc-binding modules of the LIM domains, despite limited sequence identity, lead to the proposal of a single zinc-binding motif in LIM domains. The coordinates are available from the Brookhaven protein data bank, entry 1ZFO.
The primary and secondary structure of glutaredoxin-3 (Grx3), a glutathione-disulfide oxidoreductase from Escherichia coli, has been determined. The amino acid sequence of Grx3 consists of 82 residues and contains a redox-active motif, Cys-Pro-Tyr-Cys, typical of the glutaredoxin family. Sequence comparison reveals a homology (33% identity) to that of glutaredoxin-1 (Grx1) from E. coli as well as to other members of the thioredoxin superfamily. In addition to the active site cysteine residues, Grx3 contains one additional cysteine (Cys65) corresponding to one of the two non-active site (or structural) cysteine residues present in mammalian glutaredoxins. The sequence-specific 1H and 15N nuclear magnetic resonance assignments of reduced Grx3 have been obtained. From a combined analysis of chemical shifts, 3JHNalpha coupling constants, sequential and medium range NOEs, and amide proton exchange rates, the secondary structure of reduced Grx3 was determined and found to be very similar to that inferred from amino acid sequence comparison to homologous proteins. The consequences of the proposed structural similarity to Grx1 are that Grx3, while possessing a largely intact GSH binding cleft, would have a very different spatial distribution of charged residues, most notably surrounding the active site cysteine residues and occurring in the proposed hydrophobic protein-protein interaction area. These differences may contribute to the observed very low Kcat of Grx3 as a reductant of insulin disulfides or as a hydrogen donor for ribonucleotide reductase. Thus, despite an identical active site disulfide motif and a similar secondary structure and tertiary fold, Grx3 and Grx1 display large functional differences in in vitro protein disulfide oxido-reduction reactions.
The proton exchange rates between water and the hydroxyl protons of threonine, serine, tyrosine, the amino protons of lysine, and the guanidinium protons of arginine were measured in the pH range 0.5 to 8.5 and for the temperatures 4 degrees C, 10 degrees C, 20 degrees C, 30 degrees C, and 36 degrees C. The intrinsic exchange rates of the hydroxyl and amino protons at pH 7.0 degrees C and 36 degrees C were found to be in the range 700 to about 10,000 s-1. In addition, the exchange catalysis by phosphate, carbonate, carboxyl-, and amino-groups was investigated. The presence of these exchange catalysts at physiological concentrations increased the proton exchange rates from hydroxyl and amino groups several fold. The proton exchange rates are sufficiently fast that the total magnetization transfer between biomolecules and free bulk water is not rate limited by the proton exchange rate, but by the intramolecular cross-relaxation rates between the exchangeable and nonexchangeable protons of the biomolecules. Since the cross-relaxation rates between surface hydration water molecules and biomolecules are usually vanishingly small because of too rapid exchange with the free bulk water, it is proposed that the contrast in MR images is a fingerprint of the number of the exchangeable protons from OH and NH groups of the tissue, as far as the contrast depends on the magnetization transfer between biomolecules and water.
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Virtually complete sequence specific 1H and 15N resonance assignments are presented for acid denatured reduced E. coli glutaredoxin 3. The sequential resonance assignments of the backbone rely on the combined use of 3D F1-decoupled ROESY-15N-HSQC and 3D 15N-HSQC-(TOCSY-NOESY)-15N-HSQC using a single uniformly 15N labelled protein sample. The sidechain resonances were assigned from a 3D TOCSY-15N-HSQC and a homonuclear TOCSY spectrum. The presented assignment strategy works in the absence of chemical exchange peaks with signals from the native conformation and without 13C/15N double labelling. Chemical shifts, 3J(alpha H, NH) coupling constants and NOEs indicate extensive conformational averaging of both backbone and side chains in agreement with a random coil conformation. The only secondary structure element persisting at pH 3.5 appears to be a short helical segment comprising residues 37 to 40.
During the reduction of ribonucleotides with [3H]formate by the class III anaerobic ribonucleotide reductase from Escherichia coli tritium appears in water and not in the product deoxyribonucleotide. In D2O, deuterium replaces the OH-group at carbon-2' with retention of configuration. In addition we find 1-2% deuterium in the 3'-position demonstrating a small exchange of this hydrogen with the protons of water during catalysis. Class I and II enzymes catalyze identical reactions. Members of the three classes of reductases apparently use the same chemical mechanism in spite of having completely different protein structures.
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The residence times of the hydration water molecules near the base protons of d-(GTGGAATTCCAC)2 and d-(GTGGTTAACCAC)2 were investigated by nuclear magnetic resonance (NMR) spectroscopy. Nuclear Overhauser effects (NOE) were observed between base protons of the DNA and hydration water in NOESY and ROESY experiments. Large positive NOESY cross peaks observed between the resonances of the water and the adenine 2H protons of the central d-(AATT)2 segment in the duplex d-(GTGGAATTCCAC)2 indicate the presence of a 'spine of hydration' with water molecules exhibiting residence times on the DNA longer than 1 nanosecond. In contrast, no positive intermolecular NOESY cross peaks were detected in the d-(TTAA)2 segment of the duplex d-(GTGGTTAACCAC)2, indicating that no water molecules bound with similarly long residence times occur in the minor groove of this fragment. These results can be correlated with the larger width of the minor groove in d-(TTAA)2 segments as compared to that in d-(AATT)2 segments, as observed previously in single crystal structures of related oligonucleotide duplexes in B type conformation. The present experiments confirm earlier experimental results from single crystal studies and theoretical predictions that a 5'-dTA-3' step in the nucleotide sequence interrupts the spine of hydration in the minor groove.
The three-dimensional solution structure of porcine PEC-60, a 60 amino acid residue protein of the Kazal type family of proteinase inhibitors, was determined by nuclear magnetic resonance (NMR) spectroscopy. The structure determination is based on nearly complete 1H, 13C and 15N resonance assignments including stereospecific 1H resonance assignments for 40 pairs of methylene protons and isopropyl methyl groups. The stereospecific resonance assignments of the beta-protons were supported by heteronuclear long-range correlation experiments recorded at natural 13C and 15N isotopic abundances. A group of 20 conformers were calculated using the experimentally derived NMR constraints with the program DIANA, and energy-minimized in a 4 A water shell using the program OPAL. The average of the root-mean-square deviations relative to the mean structure of the 20 conformers selected to represent the solution structure of PEC-60 is 0.55 A for the backbone atoms of residues 6 to 10 and 24 to 60. Disordered conformations are observed for the amino-terminal pentapeptide and the polypeptide segment containing residues 11 to 23. The NMR structure confirms the structural similarity of PEC-60 to the Kazal type family of proteinase inhibitors which had been previously suggested on the basis of amino acid homology. The well-defined part of PEC-60 contains a short three-stranded anti-parallel beta-sheet involving the residues 27 to 29, 33 to 35 and 53 to 56 with a beta-bulge at residue 55, a type I turn comprising residues 29 to 32, and an alpha-helix involving the residues 37 to 48. T1(13C) relaxation measurements of the alpha-carbons and linewidth measurements of the amide proton signals indicate substantially increased mobility on the pico- to nanosecond time-scale for the amino-terminal pentapeptide as well as within the loop comprising residues 11 to 23. The structure of PEC-60 is compared to the X-ray crystal structures of homologous Kazal type proteinase inhibitors and the dynamic properties of PEC-60 are discussed with respect to the observed lack of any substantial trypsin inhibiting activity.
The solution structure of the N-terminal DNA-binding domain of the P22 c2 repressor (residues 1 to 76) was determined by nuclear magnetic resonance (NMR) spectroscopy. The structure determination was based on nearly complete sequence-specific resonance assignments for 1H, 13C and 15N, and tables of the chemical shifts for all three nuclei are included here. A group of 20 conformers was calculated from the NMR constraints using the program DIANA, and energy-minimized using an implementation of the AMBER force field in the program OPAL. The core of the protein formed by residues 5 to 68 is structurally well defined, with an average of 0.7 A for the root-mean-square deviations calculated for the backbone atoms of the individual conformers relative to the mean coordinates. The N-terminal tetrapeptide segment and the C-terminal octapeptide segment are flexibly disordered. The molecular architecture includes five alpha-helical segments with residues 6 to 17, 21 to 28, 32 to 39, 47 to 57 and 61 to 65. The length and relative orientation of these helices are closely similar to the arrangement of corresponding regular secondary structures in the DNA-binding domain of the 434 repressor, with the sole exception of the fourth helix, which is one turn longer at its amino-terminal end than the corresponding helix in the 434 repressor. This extension of the fourth helix implies that the DNA-binding mode of the P22 c2 repressor must be somewhat different from that observed for the 434 repressor. Exact superposition of two P22 c2 repressor DNA-binding domains for best fit of corresponding polypeptide backbone atoms onto the two 434 repressor DNA-binding domains in the crystal structure of the 434 repressor-DNA complex would result in a model of the P22 c2 repressor-DNA complex which could not accommodate the fourth helices because of steric overlap.
A 1:1 complex formed by the mutant Antennapedia(C39S) homeodomain and a 14 base-pair DNA duplex (molecular weight approximately 18,000) was prepared in which the protein was uniformly 13C-labeled. Using two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy with 13C(omega 1, omega 2)-double-half-filter and three-dimensional 13C-correlated NOE spectroscopy, nearly complete sequence-specific resonance assignments were obtained for both the protein and the DNA in the complex. On this basis conformational constraints needed for a three-dimensional structure determination were collected. Using 855 intramolecular distance constraints as input, the structure of the DNA-bound Antp(C39S) homeodomain was calculated with the program DIANA, followed by restrained energy minimization with the program OPAL. A group of 20 conformers characterizes a well-defined structure for residues 8 to 56, with an average of 0.5 A of the pairwise root-mean-square deviations calculated for the backbone atoms of the individual conformers relative to the mean coordinates. The quality of the resulting structure is comparable to the one for the free protein, and the global fold of the free Antp(C39S) homeodomain was found to be conserved in the DNA complex. The structure of the DNA-bound protein was the starting point for the subsequent structure determination of the complete Antp(C39S) homeodomain-DNA complex in solution.
The nuclear magnetic resonance (NMR) solution structure of a complex formed by the mutant Antennapedia homeodomain with Cys39 replaced by Ser, Antp(C39S), and a 14 base-pair DNA duplex containing the BS2 operator sequence was determined using uniform 13C and 15N-labeling of the protein. Two-dimensional nuclear Overhauser enhancement spectroscopy ([1H,1H]NOESY) with 15N(omega 2)-half-filter and 13C(omega 1, omega 2)-double-half-filter, and three-dimensional heteronuclear-correlated [1H,1H]NOESY yielded a total of 855 intramolecular NOE upper distance constraints in the homeodomain, 151 upper distance constraints within the DNA duplex, and 39 intermolecular protein-DNA upper distance constraints. These data were used as the input for the structure calculation with simulated annealing followed by molecular dynamics in a water bath and energy refinement. A group of 16 conformers was thus generated which represent the solution structure of the Antp(C39S) homeodomain-DNA complex. The new structure determination confirms the salient features reported previously from a preliminary investigation of the same complex, in particular the location of the recognition helix in the major groove with the turn of the helix-turn-helix motif outside the contact area with the DNA, and the N-terminal arm of the homeodomain contacting the minor groove of the DNA. In addition, distinct amino acid side-chain-DNA contacts could be identified, and evidence was found that the invariant residue Asn51 (and possibly also Gln50) is in a slow dynamic equilibrium between two or several different DNA contact sites. The molecular dynamics calculations in a water bath yielded structures with hydration water molecules in the protein-DNA interface, which coincides with direct NMR observations of hydration waters. In the Appendix the experimental data obtained with the Antp(C39S) homeodomain-DNA complex and the techniques used for the structure calculation are evaluated using a simulated input data set derived from the X-ray crystal structure of a DNA complex with a homologous homeodomain. This study indicates that a nearly complete set of NOE upper distance constraints for the Antp(C39S) homeodomain and the protein-DNA interface was presently obtained. It further shows that the structure calculation used here yields a precise reproduction of the crystal structure from the simulated input data, and also results in hydration of the protein-DNA interface in the recalculated complex.
A 1 ns trajectory from a molecular dynamics study of 1.4 ns total length was used for a detailed analysis of the residence times of water molecules located near 227 selected bovine pancreatic trypsin inhibitor (BPTI) atoms. The simulation was performed using the GROMOS force field, with apolar hydrogen atoms treated as united atoms, and the SPC/E water model. The system consisted of 568 BPTI atoms and 2371 water molecules. The theoretical results are in good agreement with experimental data available from nuclear magnetic resonance spectroscopy. The residence times of individual water molecules coming near a given BPTI atom, as obtained from the simulation, vary greatly and range between 10 and 500 ps. The effective residence time, calculated using a correlation function technique from the presence of all individual water molecules visiting the hydration shell of a given BPTI atom, never exceeds 200 ps. The average residence time near backbone and side-chain atoms is approximately 39 ps and 24 ps, respectively. The shortest residence times, on average, are found near charged atoms (19 ps), whereas near non-polar and polar side-chain atoms the residence times are 25 ps and 36 ps, respectively. There is no apparent correlation between the residence times of the hydration water molecules of solvent-accessible residues and their location in different regular or non-regular secondary structures.
Two conformational isomers were observed in the 1H nuclear magnetic resonance (NMR) spectra of the basic pancreatic trypsin inhibitor (BPTI) and of a mutant protein with Gly 36 replaced by Ser, BPTI(G36S). The less abundant isomer differs from the major conformation by different chirality of the Cys 14-Cys 38 disulfide bond. In BPTI, the population of the minor conformer increases from about 1.5% at 4 degrees C to 8% at 68 degrees C. In BPTI(G36S), the population of the minor conformation is about 15% of the total protein, so that a detailed structural study was technically feasible; a trend toward increasing population of the minor conformer at higher temperatures was observed also for this mutant protein. The activation parameters for the exchange between the two conformations were measured in the temperature range 4-68 degrees C, using uniformly 15N-enriched protein samples. Below room temperature the exchange rate of the disulfide flip follows an Arrhenius-type temperature dependence, with negative activation entropy in both proteins. At higher temperatures the exchange rates are governed by a different set of activation parameters, which are similar to those for the ring flips of Tyr 35 about the C beta-C gamma bond. Although the equilibrium enthalpy and entropy were found to be largely temperature independent, the activation entropy changes sign and is positive at higher temperatures. These results suggest that, above room temperature, the disulfide flips are coupled to the same protein structure fluctuations as the ring flips of Tyr 35.