Three-dimensional solution structure of plastocyanin from the green alga Scenedesmus obliquus.

The solution conformation of plastocyanin from the green alga Scenedesmus obliquus has been determined from distance and dihedral angle constraints derived by nuclear magnetic resonance (NMR) spectroscopy. Structures were generated with distance geometry and restrained molecular dynamics calculations. A novel molecular replacement method was also used with the same NMR constraints to generate solution structures of S. obliquus plastocyanin from the x-ray structure of the homologous poplar protein. Scenedesmus obliquus plastocyanin in solution adopts a beta-barrel structure. The backbone conformation is well defined and is similar overall to that of poplar plastocyanin in the crystalline state. The distinctive acidic region of the higher plant plastocyanins, which functions as a binding site for electron transfer proteins and inorganic complexes, differs in both shape and charge in S. obliquus plastocyanin.

Three-dimensional solution structure of a single zinc finger DNA-binding domain.

The three-dimensional solution structure of a zinc finger nucleic acid binding motif has been determined by nuclear magnetic resonance (NMR) spectroscopy. Spectra of a synthetic peptide corresponding to a single zinc finger from the Xenopus protein Xfin yielded distance and dihedral angle constraints that were used to generate structures from distance geometry and restrained molecular dynamics calculations. The zinc finger is an independently folded domain with a compact globular structure in which the zinc atom is bound by two cysteine and two histidine ligands. The polypeptide backbone fold consists of a well-defined helix, starting as alpha and ending as 3(10) helix, packed against two beta strands that are arranged in a hairpin structure. A high density of basic and polar amino acid side chains on the exposed face of the helix are probably involved in DNA binding.

Interpretation of chemical shifts and coupling constants in macromolecules.

Recent developments in NMR spectroscopy, along with advances in computational techniques, have produced new approaches to the interpretation of chemical shifts and spin-spin coupling constants in biomolecules. Quantum chemical studies of useful accuracy are now becoming more routine and are increasingly being used in conjunction with experimental studies to map out expected structural patterns for peptides and oligonucleotides. Topics of recent special interest include spin couplings across hydrogen bonds and patterns of chemical shift anisotropies, in both diamagnetic and paramagnetic proteins.

The use of chemical shifts and their anisotropies in biomolecular structure determination.

The existence of chemical shift dispersion is crucial for the application of NMR spectroscopy to biomolecules, but the direct interpretation of shift tensors in terms of structure and dynamics is often difficult. Proton shifts reflect environmental influences from nearby aromatic groups, metal sites or hydrogen-bonding partners. These effects can be reasonably modeled with empirical equations, but multiple contributions to shifts can be difficult to disentangle. Shifts for carbon and nitrogen generally reflect local bonding interactions, often in ways that allow the local structure to be inferred. The anisotropy of the shielding tensor is also of interest. It influences the resonance position in partially-ordered samples and has consequences for spin relaxation, even in isotropic systems. There has been recent progress in measuring and interpreting these anisotropies.

RNAMotif, an RNA secondary structure definition and search algorithm.

RNA molecules fold into characteristic secondary and tertiary structures that account for their diverse functional activities. Many of these RNA structures are assembled from a collection of RNA structural motifs. These basic building blocks are used repeatedly, and in various combinations, to form different RNA types and define their unique structural and functional properties. Identification of recurring RNA structural motifs will therefore enhance our understanding of RNA structure and help associate elements of RNA structure with functional and regulatory elements. Our goal was to develop a computer program that can describe an RNA structural element of any complexity and then search any nucleotide sequence database, including the complete prokaryotic and eukaryotic genomes, for these structural elements. Here we describe in detail a new computational motif search algorithm, RNAMotif, and demonstrate its utility with some motif search examples. RNAMotif differs from other motif search tools in two important aspects: first, the structure definition language is more flexible and can specify any type of base-base interaction; second, RNAMotif provides a user controlled scoring section that can be used to add capabilities that patterns alone cannot provide.

High-resolution solution structures of oxidized and reduced Escherichia coli thioredoxin.

BACKGROUND: Thioredoxin participates in thiol-disulfide exchange reactions and both oxidized thioredoxin (disulfide form) and reduced thioredoxin (dithiol form) are found under physiological conditions. Previous structural studies suggested that the two forms were extremely similar, although significant functional and spectroscopic differences exist. We therefore undertook high-resolution solution structural studies of the two forms of Escherichia coli thioredoxin in order to detect subtle conformational differences. RESULTS: The solution structures of reduced and oxidized thioredoxin are extremely similar. Backbone structure is largely identical in the two forms, with slight differences in the region of the active site, which includes Cys32 and Cys35. The side chain sulfur atom of Cys32 is tilted away from that of Cys35 in the reduced form of the protein to accommodate the increase in S-S distance that occurs upon reduction of the disulfide, but the chi 1 angles of the two cysteines remain the same in the two forms. CONCLUSIONS: Only subtle conformational changes occur upon changing the oxidation state of the active site cysteines, including the positions of some side chains and in hydrogen bonding patterns in the active site region. Functional differences between the two forms are probably therefore related to differences in local conformational flexibility in and near the active site loop.

Small molecule hydration energy and entropy from 3D-RISM.

Implicit solvent models offer an attractive way to estimate the effects of a solvent environment on the properties of small or large solutes without the complications of explicit simulations. One common test of accuracy is to compute the free energy of transfer from gas to liquid for a variety of small molecules, since many of these values have been measured. Studies of the temperature dependence of these values (i.e. solvation enthalpies and entropies) can provide additional insights into the performance of implicit solvent models. Here, we show how to compute temperature derivatives of hydration free energies for the 3D-RISM integral equation approach. We have computed hydration free energies of 1123 small drug-like molecules (both neutral and charged). Temperature derivatives were also used to calculate hydration energies and entropies of 74 of these molecules (both neutral and charged) for which experimental data is available. While direct results have rather poor agreement with experiment, we have found that several previously proposed linear hydration free energy correction schemes give good agreement with experiment. These corrections also provide good agreement for hydration energies and entropies though simple extensions are required in some cases.

NMR and molecular dynamics studies of the hydration of a zinc finger-DNA complex.

The hydration of a high-affinity protein-DNA complex involving the three amino terminal zinc finger domains of transcription factor IIIA (TFIIIA) and a 15-base-pair DNA duplex was investigated by NMR spectroscopy and molecular dynamics (MD) simulations. Intermolecular nuclear Overhauser effects (NOEs) between protein and water provided an experimental basis for identifying potential sites of hydration. These initial assignments were evaluated with the aid of two, 2 ns MD simulations of the protein-DNA complex conducted with the explicit inclusion of water solvent. The two independent simulations produced similar trends in terms of water residence times around the solute, and these results were used to separate protein-water NOEs from alternate exchange-relayed cross peaks. Furthermore, only six of the 170 protons which failed to show intermolecular NOEs to solvent showed nearby long-resident water molecules in the MD simulations, illustrating an impressive level of agreement between theory and experiment. Analyses of the MD trajectories also allowed an examination of the role of water in recognition and binding affinity of the zinc fingers with DNA. The interface is well hydrated, characterized by direct contacts between the protein and DNA, as well as mediating water bridges. Approximately 18 water-mediated hydrogen bonds between the protein and DNA were observed on average. Roughly half of these were water molecules with long residence times that are most likely to be important for binding, since they involve residues which have been shown through biochemical studies to be crucial for protein-DNA binding. This level of atomic detail could not otherwise be established through the existing NMR and crystal structures of the TFIIIA-DNA complex.

Thermodynamics of a reverse turn motif. Solvent effects and side-chain packing.

The linear pentapeptide, Ala-Tyr-cis-Pro-Tyr-Asp-NMA (AYPYD) is known to have a significant population of type VI turn conformers in aqueous solvent. We have carried out theoretical studies of the conformational energetics of this peptide using a potential of mean force (PMF) consisting of the AMBER/OPLS empirical potential energy function, a macroscopic electrostatic model of polar solvation, and a surface area-based model of non-polar solvation. Conformers were taken from molecular dynamics simulations reported elsewhere, or generated by a random search method reported here. The chain entropy of folding was calculated by a systematic search of accessible dihedral angle space. The intra-peptide component was found to strongly favor folding and was nearly cancelled by the polar solvation term which disfavored folding. The non-polar solvation term had little effect. Fluctuations about the average value of the PMF were small and in accord with estimates from a simple harmonic model. When applied to conformers generated by a random search, the PMF selected a conformer close to the NMR-determined structure as the lowest energy conformer. The conformer with the second-lowest energy was extended, but was found to fold rapidly to the turn state in a subsequent molecular dynamics study, and may be an important state on the folding-unfolding pathway. Averages of the PMF were combined with the entropy estimates to provide an estimate of the free energy of folding that is in reasonable agreement with experimental results. In terms of the interplay between backbone electrostatic interactions and the packing of apolar side-chains, this peptide provides a model for the energetics of protein folding, and therefore makes a useful test case for calculations.

Solution structure of carbonmonoxy myoglobin determined from nuclear magnetic resonance distance and chemical shift constraints.

Solution NMR structures for sperm whale carbonmonoxy myoglobin have been calculated using 1301 distance restraints determined from nuclear Overhauser enhancement (NOE) measurements on 15N-labeled protein and chemical shift calculations for 385 protons. Starting structures included four crystal forms of myoglobin and 12 structures generated by metric matrix distance geometry. Refinements were also carried out using distance restraints alone. In general, the solution conformations are very close to the crystal structures, although the crystal structures are not consistent with some of the observed NOE connectivities. The solution structures are about as far apart from each other (as measured by backbone root-mean-square deviations) as they are from the crystal conformation. Inclusion of chemical shift restraints both tightened the spread of computed structures (especially in the heme pocket region) and led to structures that were closer to the X-ray conformation. The disposition of the side-chains near the heme group could in many cases be determined with considerable confidence, suggesting that a chemical shift analysis may be a useful adjunct to other sources of structural information available from NMR. In particular, this evidence suggests that the distal histidine residue is slightly displaced from the crystal conformation, but still inside the heme pocket at pH 5.6, that the side-chain of Leu89 is in contact with the heme ring but is probably disordered, and that the heme pocket where ligands bind is virtually identical in solution and in the crystal forms.

The structural basis for in situ activation of DNA alkylation by duocarmycin SA.

Duocarmycin SA is a member of a growing class of interesting lead compounds for chemotherapy, distinguished by the manner in which they bind to and react with DNA substrates. The first three-dimensional structure of a DNA adduct of an unnatural enantiomer from this family has been determined by (1)H NMR methods. Comparison to the previously determined structure of the natural enantiomer bound in the same DNA-binding site provides unique insights into the similarities and critical distinctions producing the respective alkylation products and site selectivities. The results also support the hypothesis that the duocarmycin SA alkylation reaction is catalyzed by the binding to DNA, and provide a deeper understanding of the structural basis for this unique mode of activation.

Solution structure of the first three zinc fingers of TFIIIA bound to the cognate DNA sequence: determinants of affinity and sequence specificity.

The high resolution solution structure of a protein containing the three amino-terminal zinc fingers of Xenopus laevis transcription factor IIIA (TFIIIA) bound to its cognate DNA duplex was determined by nuclear magnetic resonance spectroscopy. The protein, which is designated zf1-3, binds with all three fingers in the DNA major groove, with a number of amino acids making base-specific contacts. The DNA structure is close to B-form. Although the mode of interaction of zf1-3 with DNA is similar to that of zif268 and other structurally characterized zinc finger complexes, the TFIIIA complex exhibits several novel features. Each zinc finger contacts four to five base-pairs and the repertoire of known base contact residues is extended to include a tryptophan at position +2 of the helix (finger 1) and arginine at position +10 (finger 3). Sequence-specific base contacts are made over virtually the entire length of the finger 3 helix. Lysine and histidine side-chains involved in base recognition are dynamically disordered in the solution structure; in the case of lysine, in particular, this could significantly decrease the entropic cost of DNA binding. The TGEKP(N) linker sequences, which are highly flexible in the unbound protein, adopt ordered conformations on DNA binding. The linkers appear to play an active structural role in stabilization of the protein-DNA complex. Substantial protein-protein contact surfaces are formed between adjacent fingers. As a consequence of these protein-protein interactions, the orientation of finger 1 in the major groove differs from that of the other fingers. Contributions to high affinity binding by zf1-3 come from both direct protein-DNA contacts and from indirect protein-protein interactions associated with structural organization of the linkers and formation of well-packed interfaces between adjacent zinc fingers in the DNA complex. The structures provide a molecular level explanation for the large body of footprinting and mutagenesis data available for the TFIIIA-DNA complex.

High-resolution solution structure of reduced French bean plastocyanin and comparison with the crystal structure of poplar plastocyanin.

The three-dimensional solution structure of reduced (CuI) plastocyanin from French bean leaves has been determined by distance geometry and restrained molecular dynamics methods using constraints obtained from 1H n.m.r. (nuclear magnetic resonance) spectroscopy. A total of 1244 experimental constraints were used, including 1120 distance constraints, 103 dihedral angle constraints and 21 hydrogen bond constraints. Stereospecific assignments were made for 26 methylene groups and the methyls of 11 valines. Additional constraints on copper co-ordination were included in the restrained dynamics calculations. The structures are well defined with average atomic root-mean-square deviations from the mean of 0.45 A for all backbone heavy atoms and 1.08 A for side-chain heavy atoms. French bean plastocyanin adopts a beta-sandwich structure in solution that is similar to the X-ray structure of reduced poplar plastocyanin; the average atomic root-mean-square difference between 16 n.m.r. structures and the X-ray structure is 0.76 A for all backbone heavy atoms. The conformations of the side-chains that constitute the hydrophobic core of French bean plastocyanin are very well defined. Of 47 conserved residues that populate a single chi 1 angle in solution, 43 have the same rotamer in the X-ray structure. Many surface side-chains adopt highly preferred conformations in solution, although the 3J alpha beta coupling constants often indicate some degree of conformational averaging. Some surface side-chains are disordered in both the solution and crystal structures of plastocyanin. There is a striking correlation between measures of side-chain disorder in solution and side-chain temperature factors in the X-ray structure. Side-chains that form a distinctive acidic surface region, believed to be important in binding other electron transfer proteins, appear to be disordered. Fifty backbone amide protons form hydrogen bonds to carbonyls in more than 60% of the n.m.r. structures; 45 of these amide protons exchange slowly with solvent deuterons. Ten hydrogen bonds are formed between side-chain and backbone atoms, eight of which are correlated with decreased proton exchange. Of the 60 hydrogen bonds formed in French bean plastocyanin, 56 occur in the X-ray structure of the poplar protein; two of the missing hydrogen bonds are absent as a result of mutations. It appears that molecular dynamics refinement of highly constrained n.m.r. structures allows accurate prediction of the pattern of hydrogen bonding.

Computational studies of the interaction of myoglobin and xenon.

Computational studies are used to investigate the energies of xenon binding to myoglobin and to describe pathways through the protein interior for a metmyoglobin-xenon complex. Empirical energy calculations indicate a favorable enthalpic contribution of 0.6 to 4.2 kcal/mol to xenon binding for four experimentally determined xenon sites. These calculated enthalpies help to explain the different xenon occupancies observed experimentally. A fifth site, modeled in place of the iron co-ordinated water molecule in the distal cavity, is also predicted to bind xenon. The largest contribution to the binding energy is from van der Waals' interactions with smaller contributions from polarization and protein strain terms. Ligand trajectory calculations as well as a new geometric algorithm define a connecting network of channel-like pathways through the static protein structure. One or two pathways appear to lead most easily from each major internal cavity to the protein surface. The importance of these channels in protein dynamics and their implications as routes for ligand motion are discussed.

NMR solution structure of Cu(I) rusticyanin from Thiobacillus ferrooxidans: structural basis for the extreme acid stability and redox potential.

The solution structure of the Cu(I) form of the rusticyanin from Thiobacillus ferrooxidans has been calculated from a total of 1979 distance and dihedral angle constraints derived from 1H, 13C and 15N NMR spectra. The structures reveal two beta-sheets, one of six strands and one of seven strands that are tightly packed in a beta-barrel or beta-sandwich arrangement, and a short helix that extends on the outside of one of the sheets to form a second hydrophobic core. The copper coordination sphere is composed of the standard type I ligands (His2CysMet) in a distorted tetrahedral arrangement. The copper-binding site is located within a hydrophobic region at one end of the molecule, surrounded by a number of aromatic rings and hydrophobic residues. This configuration probably contributes to the acid stability of the copper site, since close association of the aromatic rings with the histidine ligands would sterically hinder their dissociation from the copper. An electrostatic analysis based on a comparison of the structures of rusticyanin and French bean plastocyanin shows that factors determining the high redox potential of rusticyanin include contributions from charged side-chains and from the disposition of backbone peptide dipoles, particularly in the 81 to 86 region of the sequence and the ligand cysteine residue. These interactions should also contribute to the acid stability by inhibiting protonation of His143.

NMR solution structure of the inserted domain of human leukocyte function associated antigen-1.

The interaction between the leukocyte function-associated antigen-1 (LFA-1) and the intercellular adhesion molecule is thought to be mediated primarily via the inserted domain (I-domain) in the alpha-subunit. The activation of LFA-1 is an early step in triggering the adhesion of leukocytes to target cells decorated with intercellular adhesion molecules. There is some disagreement in the literature over the respective roles of conformational changes in the I-domain and of divalent cations (Mg(2+), Mn(2+)) in the activation of LFA-1 for intercellular adhesion molecule binding. X-ray crystallographic structures of the I-domains of LFA-1 and Mac-1 in the presence and absence of cations show structural differences in the C-terminal alpha-helix; this change was proposed to represent the active and inactive conformations of the I-domain. However, more recent X-ray results have called this proposal into question. The solution structure of the Mg(2+) complex of the I-domain of LFA-1 has been determined by NMR methods, using a model-based approach to nuclear Overhauser enhancement spectroscopy peak assignment. The protein adopts the same structure in solution as that of the published I-domain X-ray structures, but the C-terminal region, where the X-ray structures are most different from each other, is different again in the solution structures. The secondary structure of this helix is well formed, but NMR relaxation data indicate that there is considerable flexibility present, probably consisting of breathing or segmental motion of the helix. The conformational diversity seen in the various X-ray structures could be explained as a result of the inherent flexibility of this C-terminal region and as a result of crystal contacts. Our NMR data are consistent with a model where the C-terminal helix has the potential flexibility to take up alternative conformations, for example, in the presence and absence of the intercellular adhesion molecule ligand. The role of divalent cations appears from our results not to be as a direct mediator of a conformational change that alters affinity for the ligand. Rather, the presence of the cation appears to be involved in some other way in ligand binding, perhaps by acting as a bridge to the ligand and by modulation of the charge of the binding surface.

High resolution solution structure of a DNA duplex alkylated by the antitumor agent duocarmycin SA.

The three-dimensional solution structure of duocarmycin SA in complex with d-(G1ACTAATTGAC11).d-(G12TCATTAGTC22) has been determined by restrained molecular dynamics and relaxation matrix calculations using experimental NOE distance and torsion angle constraints derived from 1H NMR spectroscopy. The final input data consisted of a total of 858 distance and 189 dihedral angle constraints, an average of 46 constraints per residue. In the ensemble of 20 final structures, there were no distance constraint violations >0.06 A or torsion angle violations >0.8 degrees. The average pairwise root mean square deviation (RMSD) over all 20 structures for the binding site region is 0.57 A (average RMSD from the mean: 0.39 A). Although the DNA is very B-like, the sugar-phosphate backbone torsion angles beta, epsilon, and zeta are distorted from standard values in the binding site region. The structure reveals site-specific bonding of duocarmycin SA at the N3 position of adenine 19 in the AT-rich minor groove of the duplex and binding stabilization via hydrophobic interactions. Comparisons have been made to the structure of a closely related complex of duocarmycin A bound to an AT-rich DNA duplex. These results provide insights into critical aspects of the alkylation site selectivity and source of catalysis of the DNA alkylating agents, and the unusual stability of the resulting adducts.

Solution structure and dynamics of yeast elongin C in complex with a von Hippel-Lindau peptide.

Elongin is a transcription elongation factor that stimulates the rate of elongation by suppressing transient pausing by RNA polymerase II at many sites along the DNA. It is heterotrimeric in mammals, consisting of elongins A, B and C subunits, and bears overall similarity to a class of E3 ubiquitin ligases known as SCF (Skp1-Cdc53 (cullin)-F-box) complexes. A subcomplex of elongins B and C is a target for negative regulation by the von Hippel-Lindau (VHL) tumor-suppressor protein. Elongin C from Saccharomyces cerevisiae, Elc1, exhibits high sequence similarity to mammalian elongin C. Using NMR spectroscopy we have determined the three-dimensional structure of Elc1 in complex with a human VHL peptide, VHL(157-171), representing the major Elc1 binding site. The bound VHL peptide is entirely helical. Elc1 utilizes two C-terminal helices and an intervening loop to form a binding groove that fits VHL(157-171). Chemical shift perturbation and dynamics analyses reveal that a global conformational change accompanies Elc1/VHL(157-171) complex formation. Moreover, the disappearance of conformational exchange phenomena on the microsecond to millisecond time scale within Elc1 upon VHL peptide binding suggests a role for slow internal motions in ligand recognition.

Calculations of proton-binding thermodynamics in proteins.

Computational models of proton binding can range from the chemically complex and statistically simple (as in the quantum calculations) to the chemically simple and statistically complex. Much progress has been made in the multiple-site titration problem. Calculations have improved with the inclusion of more flexibility in regard to both the geometry of the proton binding and the larger scale protein motions associated with titration. This article concentrated on the principles of current calculations, but did not attempt to survey their quantitative performance. This is (1) because such comparisons are given in the cited papers and (2) because continued developments in understanding conformational flexibility and interaction energies will be needed to develop robust methods with strong predictive power. Nevertheless, the advances achieved over the past few years should not be underestimated: serious calculations of protonation behavior and its coupling to conformational change can now be confidently pursued against a backdrop of increasing understanding of the strengths and limitations of such models. It is hoped that such theoretical advances will also spur renewed experimental interest in measuring both overall titration curves and individual pKa values or pKa shifts. Exploration of the shapes of individual titration curves (as measured by Hill coefficients and other parameters) would also be useful in assessing the accuracy of computations and in drawing connections to functional behavior.

Computational methods for determining protein structures from NMR data.

The general procedures by which solution structures of proteins may be deduced from distance and angular constraints derived from NMR are reviewed, with an emphasis on practical aspects of the calculations. In addition, novel methods based on chemical shift calculations and on quantitative fits to nuclear Overhauser effect intensities are presented; these should provide improved understanding of the limits of our ability to simulate complex spectra, and may permit higher precision structures to be determined.