Loop Backbone Research Articles

Long loop prediction using the protein local optimization program

We have developed an improved sampling algorithm and energy model for protein loop prediction, the combination of which has yielded the first methodology capable of achieving good results for the prediction of loop backbone conformations of 11 residue length or greater. Applied to our newly constructed test suite of 104 loops ranging from 11 to 13 residues, our method obtains average/median global backbone root-mean-square deviations (RMSDs) to the native structure (superimposing the body of the protein, not the loop itself) of 1.00/0.62 A for 11 residue loops, 1.15/0.60 A for 12 residue loops, and 1.25/0.76 A for 13 residue loops. Sampling errors are virtually eliminated, while energy errors leading to large backbone RMSDs are very infrequent compared to any previously reported efforts, including our own previous study. We attribute this success to both an improved sampling algorithm and, more critically, the inclusion of a hydrophobic term, which appears to approximately fix a major flaw in SGB solvation model that we have been employing. A discussion of these results in the context of the general question of the accuracy of continuum solvation models is presented.

Homology modeling using parametric alignment ensemble generation with consensus and energy-based model selection

The accuracy of a homology model based on the structure of a distant relative or other topologically equivalent protein is primarily limited by the quality of the alignment. Here we describe a systematic approach for sequence-to-structure alignment, called ‘K*Sync’, in which alignments are generated by dynamic programming using a scoring function that combines information on many protein features, including a novel measure of how obligate a sequence region is to the protein fold. By systematically varying the weights on the different features that contribute to the alignment score, we generate very large ensembles of diverse alignments, each optimal under a particular constellation of weights. We investigate a variety of approaches to select the best models from the ensemble, including consensus of the alignments, a hydrophobic burial measure, low- and high-resolution energy functions, and combinations of these evaluation methods. The effect on model quality and selection resulting from loop modeling and backbone optimization is also studied. The performance of the method on a benchmark set is reported and shows the approach to be effective at both generating and selecting accurate alignments. The method serves as the foundation of the homology modeling module in the Robetta server.

Minimalist explicit solvation models for surface loops in proteins.

We have performed molecular dynamics simulations of protein surface loops solvated by explicit water, where a prime focus of the study is the small numbers (e.g., ~100) of explicit water molecules employed. The models include only part of the protein (typically 500 - 1000 atoms), and the water molecules are restricted to a region surrounding the loop. In this study, the number of water molecules (N(w)) is systematically varied, and convergence with large N(w) is monitored to reveal N(w)(min), the minimum number required for the loop to exhibit realistic (fully hydrated) behavior. We have also studied protein surface coverage, as well as diffusion and residence times for water molecules as a function of N(w). A number of other modeling parameters are also tested. These include the number of environmental protein atoms explicitly considered in the model, as well as two ways to constrain the water molecules to the vicinity of the loop (where we find one of these methods to perform better when N(w) is small). The results (for RMSD and its fluctuations for four loops) are further compared to much larger, fully solvated systems (using ~10,000 water molecules under periodic boundary conditions and Ewald electrostatics), and to results for the GBSA implicit solvation model. We find that the loop backbone can stabilize with a surprisingly small number of water molecules (as low as 5 molecules per amino acid residue). The side chains of the loop require somewhat larger N(w), where the atomic fluctuations become too small if N(w) is further reduced. Thus, in general, we find adequate hydration to occur at roughly 12 water molecules per residue. This is an important result, because at this hydration level, computational times are comparable to those required for GBSA. Therefore these "minimalist explicit models" can provide a viable and potentially more accurate alternative. The importance of protein loop modeling is discussed in the context of these, and other, loop models, along with other challenges including the relevance of appropriate free energy simulation methodology for assessment of conformational stability.

Structural basis of HIV-1 neutralization: implications for vaccine design

Antibodies that can potently neutralize a broad spectrum of HIV-1 primary isolates are extremely rare and invaluable for innovative HIV1 vaccine design strategies. Crystal structures for four of the five antibodies [b12; 2G12; 447-52D; and 4E10] have been determined. Antibody b12 interacts with the recessed CD4 binding site through a long CDR H3 loop. Anti-gp120 antibody 2g12 recognizes a cluster of high-mannose sugars on the surface of gp120—an unexpected high affinity for a carbohydrate epitope. The 2g12 Fab arms dimerize via exchange of their VH domains to form a multivalent binding surface for carbohydrates that is useful for designing a carbohydrate-based vaccine. Antibody 447-52D also uses a long CDR H3 loop, but it interacts with the V3 loop backbone of gp120, which explains its broad specificity. 4E10 is the most broadly neutralizing and is effective against all clades and subtypes of HIV-1. The 4E10 structure with a gp41 peptide shows a helical conformation for the epitope that gives insights into the membrane fusion events. Thus, these structural studies not only elucidate how each antibody interacts with its respective antigenic site in either gp120 or gp41, but also give fascinating insights into how the immune system evolves strategies to overcome challenges in accessing epitopes that are deeply-buried (b12), have low antigenicity (2g12), that vary in sequence (447-52D), and are transiently-accessible (4E10). The novel modes of antigen recognition provide a plethora of new ideas for the design of novel HIV-1 immunogens to elicit such antibody responses and are being harnessed in a retrovaccinology approach for HIV-1 vaccine design.

Structural insights into SRP RNA: An induced fit mechanism for SRP assembly

Proper assembly of large protein-RNA complexes requires sequential binding of the proteins to the RNA. The signal recognition particle (SRP) is a multiprotein-RNA complex responsible for the cotranslational targeting of proteins to biological membranes. Here we describe the crystal structure at 2.6-A resolution of the S-domain of SRP RNA from the archeon Methanococcus jannaschii. Comparison of this structure with the SRP19-bound form reveals the nature of the SRP19-induced conformational changes, which promote subsequent SRP54 attachment. These structural changes are initiated at the SRP19 binding site and transmitted through helix 6 to looped-out adenosines, which form tertiary RNA interaction with helix 8. Displacement of these adenosines enforces a conformational change of the asymmetric loop structure in helix 8. In free RNA, the three unpaired bases A195, C196, and C197 are directed toward the helical axis, whereas upon SRP19 binding the loop backbone inverts and the bases are splayed out in a conformation that resembles the SRP54-bound form. Nucleotides adjacent to the bulged nucleotides seem to be particularly important in the regulation of this loop transition. Binding of SRP19 to 7S RNA reveals an elegant mechanism of how protein-induced changes are directed through an RNA molecule and may relate to those regulating the assembly of other RNPs.

Solution structure of the ActD-5'-CCGTT3GTGG-3' complex: drug interaction with tandem G.T mismatches and hairpin loop backbone.

Binding of actinomycin D (ActD) to the seemingly single-stranded DNA (ssDNA) oligomer 5'-CCGTT3 GTGG-3' has been studied in solution using high-resolution nuclear magnetic resonance (NMR) techniques. A strong binding constant (8 x 10(6) M(-1)) and high quality NMR spectra have allowed us to determine the initial DNA structure using distance geometry as well as the final ActD-5'-CCGTT3 GTGG-3' complex structure using constrained molecular dynamics calculations. The DNA oligomer 5'-CCGTT3GTGG-3' in the complex forms a hairpin structure with tandem G.T mismatches at the stem region next to a loop of three stacked thymine bases pointing toward the major groove. Bipartite T2O-GH1 and T2O-G2NH2 hydrogen bonds were detected for the G.T mismatches that further stabilize this unusual DNA hairpin. The phenoxazone chromophore of ActD intercalates nicely between the tandem G.T mismatches in essentially one major orientation. Additional hydrophobic interactions between the ActD quinoid amino acid residues with the loop T5-T6-T7 backbone protons were also observed. The hydrophobic G-phenoxazone-G interaction in the ActD-5'-CCGTT3GTGG-3' complex is more robust than that of the classical ActD- 5'-CCGCT3GCGG-3' complex, consistent with the roughly 2-fold stronger binding of ActD to the 5'-CCGTT3GTGG-3' sequence than to its 5'-CCG CT3GCGG-3' counterpart. Stabilization by ActD of a hairpin containing non-canonical stem base pairs further strengthens the notion that ActD or other related compounds may serve as a sequence- specific ssDNA-binding agent that inhibits human immunodeficiency virus (HIV) and other retroviruses replicating through ssDNA intermediates.

Proximity of the invariant loop of U5 snRNA to the second intron residue during pre-mRNA splicing.

A photoactivatable azidophenacyl group has been introduced into seven positions in the backbone of the 11 nucleotide invariant loop of U5 snRNA. By reconstituting depleted splicing extracts with reassembled U5 snRNP particles, molecular neighbors were assessed as a function of splicing. All cross-links to the pre-mRNA mapped to the second nucleotide downstream of the 5' splice site, and formed most readily when the reactive group was at the phosphate between U5 positions 42 and 43 or 43 and 44. Both their kinetics of appearance and sensitivity to oligonucleotide inhibition suggest that these cross-links capture a late state in spliceosome assembly occurring immediately prior to the first step. A later forming, second cross-linked species is a splicing product of the first cross-link, suggesting that the U5 loop backbone maintains this position through the first step. The proximity of the U5 loop backbone to the intron's 5' end provides sufficient restrictions to develop a three-dimensional model for the arrangement of RNA components in the spliceosome during the first step of pre-mRNA splicing.

Backbone dynamics of Tet repressor alpha8intersectionalpha9 loop.

A set of single Trp mutants of class B Tet repressor (TetR), in which Trp residues are located from positions 159 to 167, has been engineered to investigate the dynamics of the loop joining the alpha-helices 8 and 9. The fluorescence anisotropy decay of most mutants can be described by the sum of three exponential components. The longest rotational correlation time, 30 ns at 10 degrees C, corresponds to the overall rotation of the protein. The shortest two components, on the subnanosecond and nanosecond time scale, are related to internal motions of the protein. The initial anisotropy, in the 0.16-0.22 range, indicates the existence of an additional ultrafast motion on the picosecond time scale. Examination of physical models for underlying motions indicates that librational motions of the Trp side chain within the rotameric chi(1) x chi(2) potential wells contribute to the picosecond depolarization process, whereas the subnanosecond and nanosecond depolarization processes are related to backbone dynamics. In the absence of inducer, the order parameters of these motions, about 0.90 and 0.80 for most positions, indicate limited flexibility of the loop backbone. Anhydrotetracycline binding to TetR induces an increased mobility of the loop on the nanosecond time scale. This suggests that entropic factors might play a role in the mechanism of allosteric transition.

Motional dynamics of the catalytic loop in OMP synthase.

In de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase catalyzes the formation of orotidine 5'-monophosphate (OMP) from orotic acid and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). The known three-dimensional structure of the dimeric enzyme from Salmonella typhimurium is similar to that of other Type I phosphoribosyltransferases (nucleotide synthases) with a solvent-exposed active site atop a Rossman-type nucleotide binding fold. The three-dimensional structure of an enzyme-inhibitor complex [Henriksen et al. (1996) Biochemistry 35, 3803-3809] indicates that one of the two identical solvent-exposed loops can descend to cover the active site of the adjacent subunit of the dimeric enzyme. Catalytically essential residues are known to reside on this loop. In the present work, sensitivity toward limited proteolysis by trypsin confirms that the loop is solvent-exposed. Protection by PRPP and, to a lesser extent, by OMP demonstrates the existence of a second, trypsin-inaccessible, loop position. Two-dimensional 1H-15N NMR relaxation experiments on [alpha-15N]histidine-labeled WT OPRTase yielded backbone 15N T1 and T2 relaxation times and 15N[1H] NOE for His-105 (a loop residue) that are characteristic of small peptides. These results document that the surface loop is highly flexible in the unliganded enzyme. Addition of a hydrolytically stable PRPP analogue to the enzyme resulted in a significant reduction of His-105 peak intensity, indicating a dramatic change in the dynamic properties of the loop backbone in the analogue-ligated enzyme. 1H NMR titrations on histidine C2 protons, coupled with 1H and 31P titrations monitoring the C1H and 5-phosphate PRPP resonances, allowed the quantitation of the rates of loop movement during product release, and relate protein motion to enzymatic catalysis. These results suggest that loop opening and PRPP release is a two-step process, whose overall rate is partially rate-limiting in the reverse pyrophosphorolysis reaction.

Homology modeling by the ICM method.

Five models have been built by the ICM method for the Comparative Modeling section of the Meeting on the Critical Assessment of Techniques for Protein Structure Prediction. The targets have homologous proteins with known three-dimensional structure with sequence identity ranging from 25 to 77%. After alignment of the target sequence with the related three-dimensional structure, the modeling procedure consists of two subproblems: side-chain prediction and loop prediction. The ICM method approaches these problems with the following steps: (1) a starting model is created based on the homologous structure with the conserved portion fixed and the nonconserved portion having standard covalent geometry and free torsion angles; (2) the Biased Probability Monte Carlo (BPMC) procedure is applied to search the subspaces of either all the nonconservative side-chain torsion angles or torsion angles in a loop backbone and surrounding side chains. A special algorithm was designed to generate low-energy loop deformations. The BPMC procedure globally optimizes the energy function consisting of ECEPP/3 and solvation energy terms. Comparison of the predictions with the NMR or crystallographic solutions reveals a high proportion of correctly predicted side chains. The loops were not correctly predicted because imprinted distortions of the backbone increased the energy of the near-native conformation and thus made the solution unrecognizable. Interestingly, the energy terms were found to be reliable and the sampling of conformational space sufficient. The implications of this finding for the strategies of future comparative modeling are discussed.

On the use of minimization from many randomly generated loop structures in modeling antibody combining sites

We describe an approach to modeling the combining sites of antibodies that is based on energy minimization from many randomly generated starting conformations. This technique combines the Random Tweak method for loop backbone generation with a combinatorial approach to selecting side-chain conformations from site-specific customized rotamer libraries. Generated structures are subjected to screening for van der Waals overlaps, and acceptable conformations are refined using energy minimization. Low-energy loop structures are then evaluated using energetic, solvation, and knowledge-based criteria. The application of a solvent model that combines the continuum electrostatic program DelPhi with a surface area model for hydrophobicity is presented. Random Tweak-based modeling is compared with other systematic conformational search methods. Results obtained for two hypervariable loops in the antibody HyHel5 are reported. Random Tweak-based modeling is combined with knowledge-based predictions and loop-search techniques into an overall multideterminantal strategy for modeling novel antibody combining sites.

Hypermodified Nucleosides of tRNA: Synthesis, Chemistry, and Structural Features of Biological Interest

Publisher Summary This chapter discusses the synthesis, chemistry, and structural features of hypermodified nucleosides of tRNA. It deals with the direct effects of hypermodification altering nucleoside base-pairing and stacking properties as a result of inherent electronic and conformational changes. A brief inspection of the anticodon loop structure of tRNA Phe suggests the possibility of some indirect effects resulting from the interactions with the anticodon loop backbone, such as steric repulsion of bulk substituents, electrostatic repulsion of ionized groups (carboxyl and phosphodiester), hydrogen bonding, and Mg 2+ binding. Lack of accurate information about the tertiary structures of different tRNAs makes the evaluation of such indirect effects rather speculative. Some information can be gleaned from the simulation studies in which hypermodified nucleosides in their crystal structures are built into the yeast tRNA Phe anticodon loop at the positions of Gm 34 and of yW37, with the assumption that at least the main structural features are preserved in all tRNAs.

Restriction or amplification of wobble recognition: The structure of 2-thio-5-methylaminomethyluridine and the interaction of odd uridines with the anticodon loop backbone

FEBS LettersVolume 94, Issue 2 p. 361-364 Full-length articleFree Access Restriction or amplification of wobble recognition The structure of 2-thio-5-methylaminomethyluridine and the interaction of odd uridines with the anticodon loop backbone W. Hillen, W. Hillen Institut für Organische Chemie und Biochemie, Technische Hochschule Darmstadt, Petersenstr. 22, D-6100 Darmstadt, FRGSearch for more papers by this authorE. Egert, E. Egert Institut für Organische Chemie und Biochemie, Technische Hochschule Darmstadt, Petersenstr. 22, D-6100 Darmstadt, FRGSearch for more papers by this authorH.J. Lindner, H.J. Lindner Institut für Organische Chemie und Biochemie, Technische Hochschule Darmstadt, Petersenstr. 22, D-6100 Darmstadt, FRGSearch for more papers by this authorH.G. Gassen, H.G. Gassen Institut für Organische Chemie und Biochemie, Technische Hochschule Darmstadt, Petersenstr. 22, D-6100 Darmstadt, FRGSearch for more papers by this author W. Hillen, W. Hillen Institut für Organische Chemie und Biochemie, Technische Hochschule Darmstadt, Petersenstr. 22, D-6100 Darmstadt, FRGSearch for more papers by this authorE. Egert, E. Egert Institut für Organische Chemie und Biochemie, Technische Hochschule Darmstadt, Petersenstr. 22, D-6100 Darmstadt, FRGSearch for more papers by this authorH.J. Lindner, H.J. Lindner Institut für Organische Chemie und Biochemie, Technische Hochschule Darmstadt, Petersenstr. 22, D-6100 Darmstadt, FRGSearch for more papers by this authorH.G. Gassen, H.G. Gassen Institut für Organische Chemie und Biochemie, Technische Hochschule Darmstadt, Petersenstr. 22, D-6100 Darmstadt, FRGSearch for more papers by this author First published: October 15, 1978 https://doi.org/10.1016/0014-5793(78)80977-6Citations: 32AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 J.A. McCloskey, S. Nishimura, Acc. Chem. Res., 10, (1977), 403– 410. 2 F.H.C. Crick, J. Mol. Biol., 19, (1966), 548– 555. 3 T. Takemoto, K. Takeishi, S. Nishimura, T. Ukita, Eur. J. Biochem., 38, (1973), 489– 496. 4 K. Murao, T. Hasegawa, H. Ishikura, Nucl. Acids Res., 3, (1976), 2851– 2860. 5 T.D. Tumaitis, B.G. Lane, Biochim. Biophys. Acta, 224, (1970), 391– 403. 6 K. Chakraburtty, A. Steinschneider, R.V. Case, A.H. Mehler, Nucl. Acids Res., 2, (1975), 2069– 2075. 7 J. Weissenbach, G. Dirheimer, Biochim. Biophys. Acta, 518, (1978), 530– 534. 8 R. Vormbrock, R. Morawietz, H.G. Gassen, Biochim. Biophys. Acta, 340, (1974), 348– 358. 9 H.M. Berman, D. Marcu, P. Narayanan, J.D. Fissekis, R.L. Lipnick, Nucl. Acids Res., 5, (1978), 893– 903. 10 W. Hillen, E. Egert, H.J. Lindner, H.G. Gassen, Biochemistry, (1978), in press 11 K. Morikawa, K. Torii, Y. Iitaka, M. Tsuboi, S. Nishimura, FEBS Lett., 48, (1974), 279– 282. 12 W. Hillen, E. Egert, H.J. Lindner, H.G. Gassen, H. Vorbrüggen, J. Carb. Nucleos. Nucleot., 5, (1978), 23– 32. 13 C.G. Quigley, N.C. Seeman, A.H. Wang, F.L. Suddath, A. Rich, Nucl. Acids Res., 2, (1975), 2329– 2341. Citing Literature Volume94, Issue2October 15, 1978Pages 361-364 ReferencesRelatedInformation