Anti-insulin antibody structure and conformation. II. Molecular dynamics with explicit solvent.

Molecular dynamics at 300 K was used as a conformation searching tool to analyze a knowledge-based structure prediction of an anti-insulin antibody. Solvation effects were modeled by packing water molecules around the antigen binding loops. Some loops underwent backbone and side-chain conformational changes during the 95-ps equilibration, and most of these new, lower potential energy conformations were stable during the subsequent 200-ps simulation. Alterations to the model include changes in the intraloop, main-chain hydrogen bonding network of loop H3, and adjustments of Tyr and Lys side chains of H3 induced by hydrogen bonding to water molecules. The structures observed during molecular dynamics support the conclusion of the previous paper that hydrogen bonding will play the dominant role in antibody-insulin recognition. Determination of the structure of the antibody by x-ray crystallography is currently being pursued to provide an experimental test of these results. The simulation appears to improve the model, but longer simulations at higher temperatures should be performed.     

Refined solution structure and backbone dynamics of the archaeal MC1 protein

The 3D structure of methanogen chromosomal protein 1 (MC1), determined with heteronuclear NMR methods, agrees with its function in terms of the shape and nature of the binding surface, whereas the 3D structure determined with homonuclear NMR does not. The structure features five loops, which show a large distribution in the ensemble of 3D structures. Evidence for the fact that this distribution signifies internal mobility on the nanosecond time scale was provided by using (15)N-relaxation and molecular dynamics simulations. Structural variations of the arm (11 residues) induced large shape anisotropy variations on the nanosecond time scale that ruled out the use of the model-free formalism to analyze the relaxation data. The backbone dynamics analysis of MC1 was achieved by comparison with 20 ns molecular dynamics trajectories. Two β-bulges showed that hydrogen bond formation correlated with ? and ψ dihedral angle transitions. These jumps were observed on the nanosecond time scale, in agreement with a large decrease in (15)N-NOE for Gly17 and Ile89. One water molecule bridging NH(Glu87) and CO(Val57) through hydrogen bonding contributed to these dynamics. Nanosecond slow motions observed in loops LP3 (35-42) and LP5 (67-77) reflected the lack of stable hydrogen bonds, whereas the other loops, LP1 (10-14), LP2 (22-24), and LP4 (50-53), were stabilized by several hydrogen bonds. Dynamics are often directly related to function. Our data strongly suggest that residues belonging to the flexible regions of MC1 could be involved in the interaction with DNA.     

Computer-assisted re-design of spectrin SH3 residue clusters

We have developed a protein design computer program, called Perla, which performs searches in sequence space to uncover optimal amino acid sequences for desired protein three-dimensional structures. Optimal sequences are localised at the minima of a sequence-structure energy landscape defined using a complex scoring function (an all-atom molecular mechanics force field plus statistical terms including entropy and solvation) measured with respect to a reference state simulating a denatured protein. Sequence choices eventually optimise side chain packing, secondary structure propensities, and hydrogen bonding and electrostatics interactions. Perla was used to re-design clusters of residues of the SH3 domain of alpha-spectrin. Several mutant proteins were produced and characterised. Some of our designed proteins have significantly higher stabilities (stability enhancements about 0.25, 0.70 and 1.0 kcal mol(-1)) than the wild-type protein. These successful protein re-designs, and similar examples found in the literature, establish the quality of the structure-based computational approach to protein design.     

Calculations on crystal packing of a flexible molecule, Leu-enkephalin

Crystal packing calculations have been carried out on a substantial number of conformations of Leu-enkephalin; namely, those obtained both from crystal structures and from energy minimizations on isolated molecules, and with and without waters of crystallization. The known crystal structures represent the most energetically stable packings found. The conformations of the enkephalin molecules in the crystal are not the most stable for an isolated molecule; i.e. intermolecular interactions force the isolated molecule to change conformation in order to achieve a small packing volume and an optimal packing energy in the crystal. It is found that the packing energy of an enkephalin molecule is a reasonably smooth function of its molecular volume in the unit cell, if structures with intermolecular hydrogen bonding are excluded, and is substantially independent of other details of the molecular conformation or of the crystal packing. Hydrogen bonding provides additional stabilization of the crystal structure, and would likely permit crystallization of the system if it is sufficiently dense. Solvent molecules further stabilize the structure when they can also provide intermolecular hydrogen bonds.     

Hydrogen bonding in helical polypeptides from molecular dynamics simulations and amide hydrogen exchange analysis: alamethicin and melittin in methanol.

Molecular dynamics simulations of ion channel peptides alamethicin and melittin, solvated in methanol at 27 degrees C, were run with either regular alpha-helical starting structures (alamethicin, 1 ns; melittin 500 ps either with or without chloride counterions), or with the x-ray crystal coordinates of alamethicin as a starting structure (1 ns). The hydrogen bond patterns and stabilities were characterized by analysis of the dynamics trajectories with specified hydrogen bond angle and distance criteria, and were compared with hydrogen bond patterns and stabilities previously determined from high-resolution NMR structural analysis and amide hydrogen exchange measurements in methanol. The two alamethicin simulations rapidly converged to a persistent hydrogen bond pattern with a high level of 3(10) hydrogen bonding involving the amide NH's of residues 3, 4, 9, 15, and 18. The 3(10) hydrogen bonds stabilizing amide NH's of residues C-terminal to P2 and P14 were previously proposed to explain their high amide exchange stabilities. The absence, or low levels of 3(10) hydrogen bonds at the N-terminus or for A15 NH, respectively, in the melittin simulations, is also consistent with interpretations from amide exchange analysis. Perturbation of helical hydrogen bonding in the residues before P14 (Aib10-P14, alamethicin; T11-P14, melittin) was characterized in both peptides by variable hydrogen bond patterns that included pi and gamma hydrogen bonds. The general agreement in hydrogen bond patterns determined in the simulations and from spectroscopic analysis indicates that with suitable conditions (including solvent composition and counterions where required), local hydrogen-bonded secondary structure in helical peptides may be predicted from dynamics simulations from alpha-helical starting structures. Each peptide, particularly alamethicin, underwent some large amplitude structural fluctuations in which several hydrogen bonds were cooperatively broken. The recovery of the persistent hydrogen bonding patterns after these fluctuations demonstrates the stability of intramolecular hydrogen-bonded secondary structure in methanol (consistent with spectroscopic observations), and is promising for simulations on extended timescales to characterize the nature of the backbone fluctuations that underlie amide exchange from isolated helical polypeptides.     

Structural determinants of the conformations of medium-sized loops in proteins

Loops are integral components of protein structures, providing links between elements of secondary structure, and in many cases contributing to catalytic and binding sites. The conformations of short loops are now understood to depend primarily on their amino acid sequences. In contrast, the structural determinants of longer loops involve hydrogen-bonding and packing interactions within the loop and with other parts of the protein. By searching solved protein structures for regions similar in main chain conformation to the antigen-binding loops in immunoglobulins, we identified medium-sized loops of similar structure in unrelated proteins, and compared the determinants of their conformations. For loops that form compact substructures the major determinant of the conformation is the formation of hydrogen bonds to inward-pointing main chain atoms. For loops that have more extended conformations, the major determinant of their structure is the packing of a particular residue or residues against the rest of the protein. The following picture emerges: Medium-sized loops of similar conformation are stabilized by similar interactions. The groups that interact with the loop have very similar spatial dispositions with respect to the loop. However, the residues that provide these interactions may arise from dissimilar parts of the protein: The conformation of the loop requires certain interactions that the protein may provide in a variety of ways.     

Reconciling the solution and X-ray structures of the villin headpiece helical subdomain: molecular dynamics simulations and double mutant cycles reveal a stabilizing cation-pi interaction

The 36-residue helical subdomain of the villin headpiece, HP36, is one of the smallest cooperatively folded proteins, folding on the microsecond time scale. The domain is an extraordinarily popular model system for both experimental and computational studies of protein folding. The structure of HP36 has been determined using X-ray crystallography and NMR spectroscopy, with the resulting structures exhibiting differences in helix packing, van der Waals contacts, and hydrogen bonding. It is important to determine the solution structure of HP36 with as much accuracy as possible since this structure is widely used as a reference for simulations and experiments. We complement the existing data by using all-atom molecular dynamics simulations with explicit solvent to evaluate which of the experimental models is the better representation of HP36 in solution. After simulation for 50 ns initiated with the NMR structure, we observed that the protein spontaneously adopts structures with a backbone conformation, core packing, and C-capping motif on the third helix that are more consistent with the crystal structure. We also examined hydrogen bonding and side chain packing interactions between D44 and R55 and between F47 and R55, respectively, which were observed in the crystal structure but not in the NMR-based solution structure. Simulations showed large fluctuations in the distance between D44 and R55, while the distance between F47 and R55 remained stable, suggesting the formation of a cation-pi interaction between those residues. Experimental double mutant cycles confirmed that the F47-R55 pair has a larger energetic coupling than the D44-R55 interaction. Overall, these combined experimental and computational studies show that the X-ray crystal structure is the better reference structure for HP36 in solution at neutral pH. Our analysis also shows how detailed molecular dynamics simulations combined with experimental validation can help bridge the gap between NMR and crystallographic methods.     

Are aromatic carbon donor hydrogen bonds linear in proteins?

Proteins fold and maintain structure through the collective contributions of a large number of weak, noncovalent interactions. The hydrogen bond is one important category of forces that acts on very short distances. As our knowledge of protein structure continues to expand, we are beginning to appreciate the role that weak carbon-donor hydrogen bonds play in structure and function. One property that differentiates hydrogen bonds from other packing forces is propensity for forming a linear donor-hydrogen-acceptor orientation. To ascertain if carbon-donor hydrogen bonds are able to direct acceptor linearity, we surveyed the geometry of interactions specifically involving aromatic sidechain ring carbons in a data set of high resolution protein structures. We found that while donor-acceptor distances for most carbon donor hydrogen bonds were tighter than expected for van der Waals packing, only the carbons of histidine showed a significant bias for linear geometry. By categorizing histidines in the data set into charged and neutral sidechains, we found only the charged subset of histidines participated in linear interactions. B3LYP/6-31G**++ level optimizations of imidazole and indole-water interactions at various fixed angles demonstrates a clear orientation dependence of hydrogen bonding capacity for both charged and neutral sidechains. We suggest that while all aromatic carbons can participate in hydrogen bonding, only charged histidines are able to overcome protein packing forces and enforce linear interactions. The implications for protein modeling and design are discussed.     

Fast evaluation of internal loops in RNA secondary structure prediction.

MOTIVATION: Though not as abundant in known biological processes as proteins, RNA molecules serve as more than mere intermediaries between DNA and proteins. Research in the last 15 years demonstrates that RNA molecules serve in many roles, including catalysis. Furthermore, RNA secondary structure prediction based on free energy rules for stacking and loop formation remains one of the few major breakthroughs in the field of structure prediction, as minimum free energy structures and related quantities can be computed with full mathematical rigor. However, with the current energy parameters, the algorithms used hitherto suffer the disadvantage of either employing heuristics that risk (though highly unlikely) missing the optimal structure or becoming prohibitively time consuming for moderate to large sequences. RESULTS: We present a new method to evaluate internal loops utilizing currently used energy rules. This method reduces the time complexity of this part of the structure prediction from O(n4) to O(n3), thus reducing the overall complexity to O(n3). Even when the size of evaluated internal loops is bounded by k (a commonly used heuristic), the method presented has a competitive edge by reducing the time complexity of internal loop evaluation from O(k2n2) to O(kn2). The method also applies to the calculation of the equilibrium partition function. AVAILABILITY: Source code for an RNA secondary structure prediction program implementing this method is available at ftp://www.ibc.wustl.edu/pub/zuker/zuker .tar.Z     

Parsing nucleic acid pseudoknotted secondary structure: algorithm and applications.

Accurate prediction of pseudoknotted nucleic acid secondary structure is an important computational challenge. Prediction algorithms based on dynamic programming aim to find a structure with minimum free energy according to some thermodynamic ("sum of loop energies") model that is implicit in the recurrences of the algorithm. However, a clear definition of what exactly are the loops in pseudoknotted structures, and their associated energies, has been lacking. In this work, we present a complete classification of loops in pseudoknotted nucleic secondary structures, and describe the Rivas and Eddy and other energy models as sum-of-loops energy models. We give a linear time algorithm for parsing a pseudoknotted secondary structure into its component loops. We give two applications of our parsing algorithm. The first is a linear time algorithm to calculate the free energy of a pseudoknotted secondary structure. This is useful for heuristic prediction algorithms, which are widely used since (pseudoknotted) RNA secondary structure prediction is NP-hard. The second application is a linear time algorithm to test the generality of the dynamic programming algorithm of Akutsu for secondary structure prediction.Together with previous work, we use this algorithm to compare the generality of state-of-the-art algorithms on real biological structures.     

Structures of N-termini of helices in proteins.

We have surveyed 393 N-termini of alpha-helices and 156 N-termini of 3(10)-helices in 85 high resolution, non-homologous protein crystal structures for N-cap side-chain rotamer preferences, hydrogen bonding patterns, and solvent accessibilities. We find very strong rotamer preferences that are unique to N-cap sites. The following rules are generally observed for N-capping in alpha-helices: Thr and Ser N-cap side chains adopt the gauche - rotamer, hydrogen bond to the N3 NH and have psi restricted to 164 +/- 8 degrees. Asp and Asn N-cap side chains either adopt the gauche - rotamer and hydrogen bond to the N3 NH with psi = 172 +/- 10 degrees, or adopt the trans rotamer and hydrogen bond to both the N2 and N3 NH groups with psi = 1-7 +/- 19 degrees. With all other N-caps, the side chain is found in the gauche + rotamer so that the side chain does not interact unfavorably with the N-terminus by blocking solvation and psi is unrestricted. An i, i + 3 hydrogen bond from N3 NH to the N-cap backbone C = O in more likely to form at the N-terminus when an unfavorable N-cap is present. In the 3(10)-helix Asn and Asp remain favorable N-caps as they can hydrogen bond to the N2 NH while in the trans rotamer; in contrast, Ser and Thr are disfavored as their preferred hydrogen bonding partner (N3 NH) is inaccessible. This suggests that Ser is the optimum choice of N-cap when alpha-helix formation is to be encouraged while 3(10)-helix formation discouraged. The strong energetic and structural preferences found for N-caps, which differ greatly from positions within helix interiors, suggest that N-caps should be treated explicitly in any consideration of helical structure in peptides or proteins.     

SMotif: a server for structural motifs in proteins

SMotif is a server that identifies important structural segments or motifs for a given protein structure(s) based on conservation of both sequential as well as important structural features such as solvent inaccessibility, secondary structural content, hydrogen bonding pattern and residue packing. This server also provides three-dimensional orientation patterns of the identified motifs in terms of inter-motif distances and torsion angles. These motifs may form the common core and therefore, can also be employed to design and rationalize protein engineering and folding experiments. AVAILABILITY: SMotif server is available via the URL http://caps.ncbs.res.in/SMotif/index.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Structure of prothrombin fragment 1 refined at 2.8 A resolution

The structure of prothrombin fragment 1, solved at 2.8 A resolution (1 A = 0.1 nm) by a combination of multiple and single isomorphous replacement methods utilizing solvent flattening, has been refined by restrained least-squares methods (R = 0.24), solvent not included, using fairly stringent restraints on the molecular geometry and individual thermal parameters. The inner kringle loop possesses significantly lower B-values than the outer loops even though the former also constitutes a surface of the folded kringle structure. This surface forms the Lys sub-site of the fibrin binding site of other kringles. The hydrogen bonding network and ion pair interactions of fragment 1 appear to maintain a compact folded structure among the various loops of the kringle structure. On the other hand, since there is only one hydrogen bond between the kringle and its preceding 30 residues, considerable flexibility is suggested for the Gla-domain consistent with its disorder in crystals. A chitobiose has been located at the Asn77 glycosylation site, but only a single N-acetyl-glucosamine is ordered at Asn101. The lysine binding site region of other kringles is not properly developed in fragment 1, accounting for its lack of Lys/fibrin affinity. Most of the conserved sequence among 11 different kringles is associated with either: (1) protecting the inner loop disulfides Cys87-127, Cys115-139 upon which the folding is based; or (2) a requirement of the lysine binding site. The remainder of the conservation is generally associated with the ten reverse turns of the folding; of these 40 residues, or about half the sequence, 14 are conserved among eight different turns. The intermolecular packing consists of infinite helical columns of fragment 1 molecules related by a crystallographic 4(3) screw axis, which are held together by van der Waals' interactions of aromatic clusters from different molecules related by a crystallographic 2-fold rotation axis.     

Computational prediction of atomic structures of helical membrane proteins aided by EM maps

Integral membrane proteins pose a major challenge for protein-structure prediction because only approximately 100 high-resolution structures are available currently, thereby impeding the development of rules or empirical potentials to predict the packing of transmembrane alpha-helices. However, when an intermediate-resolution electron microscopy (EM) map is available, it can be used to provide restraints which, in combination with a suitable computational protocol, make structure prediction feasible. In this work we present such a protocol, which proceeds in three stages: 1), generation of an ensemble of alpha-helices by flexible fitting into each of the density rods in the low-resolution EM map, spanning a range of rotational angles around the main helical axes and translational shifts along the density rods; 2), fast optimization of side chains and scoring of the resulting conformations; and 3), refinement of the lowest-scoring conformations with internal coordinate mechanics, by optimizing the van der Waals, electrostatics, hydrogen bonding, torsional, and solvation energy contributions. In addition, our method implements a penalty term through a so-called tethering map, derived from the EM map, which restrains the positions of the alpha-helices. The protocol was validated on three test cases: GpA, KcsA, and MscL.     

Ternary complex between placental lactogen and the extracellular domain of the prolactin receptor

The structure of the ternary complex between ovine placental lactogen (oPL) and the extracellular domain (ECD) of the rat prolactin receptor (rPRLR) reveals that two rPRLR ECDs bind to opposite sides of oPL with pseudo two-fold symmetry. The two oPL receptor binding sites differ significantly in their topography and electrostatic character. These binding interfaces also involve different hydrogen bonding and hydrophobic packing patterns compared to the structurally related human growth hormone (hGH)-receptor complexes. Additionally, the receptor-receptor interactions are different from those of the hGH-receptor complex. The conformational adaptability of prolactin and growth hormone receptors is evidenced by the changes in local conformations of the receptor binding loops and more global changes induced by shifts in the angular relationships between the N- and C-terminal domains, which allow the receptor to bind to the two topographically distinct sites of oPL.     

The structural basis for molecular recognition by the vitamin B 12 RNA aptamer

Previous solution structures of ligand-binding RNA aptamers have shown that molecular recognition is achieved by the folding of an initially unstructured RNA around its cognate ligand, coupling the processes of RNA folding and binding. The 3 A crystal structure of the cyanocobalamin (vitamin B12) aptamer reported here suggests a different approach to molecular recognition in which elements of RNA secondary structure combine to create a solvent-accessible docking surface for a large, complex ligand. Central to this structure is a locally folding RNA triplex, stabilized by a novel three-stranded zipper. Perpendicular stacking of a duplex on this triplex creates a cleft that functions as the vitamin B12 binding site. Complementary packing of hydrophobic surfaces, direct hydrogen bonding and dipolar interactions between the ligand and the RNA appear to contribute to binding. The nature of the interactions that stabilize complex formation and the possible uncoupling of folding and binding for this RNA suggest a strong mechanistic similarity to typical protein-ligand complexes.     

Helix capping.

Helix-capping motifs are specific patterns of hydrogen bonding and hydrophobic interactions found at or near the ends of helices in both proteins and peptides. In an alpha-helix, the first four >N-H groups and last four >C=O groups necessarily lack intrahelical hydrogen bonds. Instead, such groups are often capped by alternative hydrogen bond partners. This review enlarges our earlier hypothesis (Presta LG, Rose GD. 1988. Helix signals in proteins. Science 240:1632-1641) to include hydrophobic capping. A hydrophobic interaction that straddles the helix terminus is always associated with hydrogen-bonded capping. From a global survey among proteins of known structure, seven distinct capping motifs are identified-three at the helix N-terminus and four at the C-terminus. The consensus sequence patterns of these seven motifs, together with results from simple molecular modeling, are used to formulate useful rules of thumb for helix termination. Finally, we examine the role of helix capping as a bridge linking the conformation of secondary structure to supersecondary structure.     

Amide temperature coefficients in the protein G B1 domain

Temperature coefficients have been measured for backbone amide ¹H and ¹⁵N nuclei in the B1 domain of protein G (GB1), using temperatures in the range 283–313 K, and pH values from 2.0 to 9.0. Many nuclei display pH-dependent coefficients, which were fitted to one or two pK_a values. ¹H coefficients showed the expected behaviour, in that hydrogen-bonded amides have less negative values, but for those amides involved in strong hydrogen bonds in regular secondary structure there is a negative correlation between strength of hydrogen bond and size of temperature coefficient. The best correlation to temperature coefficient is with secondary shift, indicative of a very approximately uniform thermal expansion. The largest pH-dependent changes in coefficient are for amides in loops adjacent to sidechain hydrogen bonds rather than the amides involved directly in hydrogen bonds, indicating that the biggest determinant of the temperature coefficient is temperature-dependent loss of structure, not hydrogen bonding. Amide ¹⁵N coefficients have no clear relationship with structure.     

A left-handed 3(1) helical conformation in the Alzheimer Abeta(12-28) peptide

We show for the first time that the secondary structure of the Alzheimer beta-peptide is in a temperature-dependent equilibrium between an extended left-handed 3(1) helix and a flexible random coil conformation. Circular dichroism spectra, recorded at 0.03 mM peptide concentration, show that the equilibrium is shifted towards increasing left-handed 3(1) helix structure towards lower temperatures. High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study the Alzheimer peptide fragment Abeta(12-28) in aqueous solution at 0 degrees C and higher temperatures. NMR translation diffusion measurements show that the observed peptide is in monomeric form. The chemical shift dispersion of the amide protons increases towards lower temperatures, in agreement with the increased population of a well-ordered secondary structure. The solvent exchange rates of the amide protons at 0 degrees C and pH 4.5 vary within at least two orders of magnitude. The lowest exchange rates (0.03-0.04 min(-1)) imply that the corresponding amide protons may be involved in hydrogen bonding with neighboring side chains.