Piotr Rotkiewicz

Fast procedure for reconstruction of full-atom protein models from reduced representations

We introduce PULCHRA, a fast and robust method for the reconstruction of full‐atom protein models starting from a reduced protein representation. The algorithm is particularly suitable as an intermediate step between coarse‐grained model‐based structure prediction and applications requiring an all‐atom structure, such as molecular dynamics, protein‐ligand docking, structure‐based function prediction, or assessment of quality of the predicted structure. The accuracy of the method was tested on a set of high‐resolution crystallographic structures as well as on a set of low‐resolution protein decoys generated by a protein structure prediction algorithm TASSER. The method is implemented as a standalone program that is available for download from http://cssb.biology.gatech.edu/skolnick/files/PULCHRA.

Ab initio folding of proteins using restraints derived from evolutionary information.

We present our predictions in the ab initio structure prediction category of CASP3. Eleven targets were folded, using a method based on a Monte Carlo search driven by secondary and tertiary restraints derived from multiple sequence alignments. Our results can be qualitatively summarized as follows: The global fold can be considered “correct” for targets 65 and 74, “almost correct” for targets 64, 75, and 77, “half‐correct” for target 79, and “wrong” for targets 52, 56, 59, and 63. Target 72 has not yet been solved experimentally. On average, for small helical and alpha/beta proteins (on the order of 110 residues or smaller), the method predicted low resolution structures with a reasonably good prediction of the global topology. Most encouraging is that in some situations, such as with target 75 and, particularly, target 77, the method can predict a substantial portion of a rare or even a novel fold. However, the current method still fails on some beta proteins, proteins over the 110‐residue threshold, and sequences in which only a poor multiple sequence alignment can be built. On the other hand, for small proteins, the method gives results of quality at least similar to that of threading, with the advantage of not being restricted to known folds in the protein database. Overall, these results indicate that some progress has been made on the ab initio protein folding problem. Detailed information about our results can be obtained by connecting to http://www.bioinformatics.danforthcenter.org/CASP3. Proteins Suppl 1999;3:177–185.

Generalized comparative modeling (GENECOMP): a combination of sequence comparison, threading, and lattice modeling for protein structure prediction and refinement.

An improved generalized comparative modeling method, GENECOMP, for the refinement of threading models is developed and validated on the Fischer database of 68 probe–template pairs, a standard benchmark used to evaluate threading approaches. The basic idea is to perform ab initio folding using a lattice protein model, SICHO, near the template provided by the new threading algorithm PROSPECTOR. PROSPECTOR also provides predicted contacts and secondary structure for the template‐aligned regions, and possibly for the unaligned regions by garnering additional information from other top‐scoring threaded structures. Since the lowest‐energy structure generated by the simulations is not necessarily the best structure, we employed two structure‐selection protocols: distance geometry and clustering. In general, clustering is found to generate somewhat better quality structures in 38 of 68 cases. When applied to the Fischer database, the protocol does no harm and in a significant number of cases improves upon the initial threading model, sometimes dramatically. The procedure is readily automated and can be implemented on a genomic scale. Proteins 2001;44:133–149.

Protein fragment reconstruction using various modeling techniques

Recently developed reduced models of proteins with knowledge-based force fields have been applied to a specific case of comparative modeling. From twenty high resolution protein structures of various structural classes, significant fragments of their chains have been removed and treated as unknown. The remaining portions of the structures were treated as fixed – i.e., as templates with an exact alignment. Then, the missed fragments were reconstructed using several modeling tools. These included three reduced types of protein models: the lattice SICHO (Side Chain Only) model, the lattice CABS (Cα + Cβ + Side group) model and an off-lattice model similar to the CABS model and called REFINER. The obtained reduced models were compared with more standard comparative modeling tools such as MODELLER and the SWISS-MODEL server. The reduced model results are qualitatively better for the higher resolution lattice models, clearly suggesting that these are now mature, competitive and complementary (in the range of sparse alignments) to the classical tools of comparative modeling. Comparison between the various reduced models strongly suggests that the essential ingredient for the sucessful and accurate modeling of protein structures is not the representation of conformational space (lattice, off-lattice, all-atom) but, rather, the specificity of the force fields used and, perhaps, the sampling techniques employed. These conclusions are encouraging for the future application of the fast reduced models in comparative modeling on a genomic scale.

Accurate reconstruction of all-atom protein representations from side-chain-based low-resolution models

A procedure for the reconstruction of all‐atom protein structures from side‐chain center‐based low‐resolution models is introduced and applied to a set of test proteins with high‐resolution X‐ray structures. The accuracy of the rebuilt all‐atom models is measured by root mean square deviations to the corresponding X‐ray structures and percentages of correct χ1 and χ2 side‐chain dihedrals. The benefit of including Cα positions in the low‐resolution model is examined, and the effect of lattice‐based models on the reconstruction accuracy is discussed. Programs and scripts implementing the reconstruction procedure are made available through the NIH research resource for Multiscale Modeling Tools in Structural Biology (http://mmtsb.scripps.edu). Proteins 2000;41:86–97.

Ab initio protein structure prediction via a combination of threading, lattice folding, clustering, and structure refinement.

A combination of sequence comparison, threading, lattice, and off‐lattice Monte Carlo (MC) simulations and clustering of MC trajectories was used to predict the structure of all (but one) targets of the CASP4 experiment on protein structure prediction. Although this method is automated and is operationally the same regardless of the level of uniqueness of the query proteins, here we focus on the more difficult targets at the border of the fold recognition and new fold categories. For a few targets (T0110 is probably the best example), the ab initio method produced more accurate models than models obtained by the fold recognition techniques. For the most difficult targets from the new fold categories, substantial fragments of structures have been correctly predicted. Possible improvements of the method are briefly discussed. Proteins 2001;Suppl 5:149–156.

DNA Vaccine Expressing the Mimotope of GD2 Ganglioside Induces Protective GD2 Cross-reactive Antibody Responses

The GD2 ganglioside expressed on neuroectodermally derived tumors, including neuroblastoma and melanoma, is weakly immunogenic in tumor-bearing patients and induces predominantly immunoglobulin (Ig)-M antibody responses in the immunized host. Here, we investigated whether interconversion of GD2 into a peptide mimetic form would induce GD2 cross-reactive IgG antibody responses in mice. Screening of the X(15) phage display peptide library with the anti-GD2 monoclonal antibody (mAb) 14G2a led to isolation of mimetic peptide 47, which inhibited the binding of 14G2a antibody to GD2-positive tumor cells. The peptide was also recognized by GD2-specific serum antibodies from a patient with neuroblastoma, suggesting that it bears an internal image of GD2 ganglioside expressed on the tumor cells. The molecular basis for antigenicity of the GD2 mimetic peptide, established by molecular modeling and mutagenesis studies, led to the generation of a 47-LDA mutant with an increased mimicry to GD2. Immunization of mice with peptide 47-LDA-encoded plasmid DNA elicited GD2 cross-reactive IgG antibody responses, which were increased on subsequent boost with GD2 ganglioside. The vaccine-induced antibodies recognized GD2-positive tumor cells, mediated complement-dependent cytotoxicity, and exhibited protection against s.c. human GD2-positive melanoma growth in the severe combined immunodeficient mouse xenograft model. The results from our studies provide insights into approaches for boosting GD2 cross-reactive IgG antibody responses by minigene vaccination with a protective epitope of GD2 ganglioside.

A method for the improvement of threading-based protein models

A new method for the homology‐based modeling of protein three‐dimensional structures is proposed and evaluated. The alignment of a query sequence to a structural template produced by threading algorithms usually produces low‐resolution molecular models. The proposed method attempts to improve these models. In the first stage, a high‐coordination lattice approximation of the query protein fold is built by suitable tracking of the incomplete alignment of the structural template and connection of the alignment gaps. These initial lattice folds are very similar to the structures resulting from standard molecular modeling protocols. Then, a Monte Carlo simulated annealing procedure is used to refine the initial structure. The process is controlled by the models internal force field and a set of loosely defined restraints that keep the lattice chain in the vicinity of the template conformation. The internal force field consists of several knowledge‐based statistical potentials that are enhanced by a proper analysis of multiple sequence alignments. The template restraints are implemented such that the model chain can slide along the template structure or even ignore a substantial fraction of the initial alignment. The resulting lattice models are, in most cases, closer (sometimes much closer) to the target structure than the initial threading‐based models. All atom models could easily be built from the lattice chains. The method is illustrated on 12 examples of target/template pairs whose initial threading alignments are of varying quality. Possible applications of the proposed method for use in protein function annotation are briefly discussed. Proteins 1999;37:592–610. ©1999 Wiley‐Liss, Inc.

Structural and functional analysis of the globular head domain of p115 provides insight into membrane tethering.

Molecular tethers have a central role in the organization of the complex membrane architecture of eukaryotic cells. p115 is a ubiquitous, essential tether involved in vesicle transport and the structural organization of the exocytic pathway. We describe two crystal structures of the N-terminal domain of p115 at 2.0 A resolution. The p115 structures show a novel alpha-solenoid architecture constructed of 12 armadillo-like, tether-repeat, alpha-helical tripod motifs. We find that the H1 TR binds the Rab1 GTPase involved in endoplasmic reticulum to Golgi transport. Mutation of the H1 motif results in the dominant negative inhibition of endoplasmic reticulum to Golgi trafficking. We propose that the H1 helical tripod contributes to the assembly of Rab-dependent complexes responsible for the tether and SNARE-dependent fusion of membranes.

Model of three-dimensional structure of vitamin D receptor and its binding mechanism with 1alpha,25-dihydroxyvitamin D(3).

Comparative modeling of the vitamin D receptor three‐dimensional structure and computational docking of 1α,25‐dihydroxyvitamin D3 into the putative binding pocket of the two deletion mutant receptors: (207–423) and (120–422, Δ [164–207]) are reported and evaluated in the context of extensive mutagenic analysis and crystal structure of holo hVDR deletion protein published recently. The obtained molecular model agrees well with the experimentally determined structure. Six different conformers of 1α,25‐dihydroxyvitamin D3 were used to study flexible docking to the receptor. On the basis of values of conformational energy of various complexes and their consistency with functional activity, it appears that 1α,25‐dihydroxyvitamin D3 binds the receptor in its 6‐s‐trans form. The two lowest energy complexes obtained from docking the hormone into the deletion protein (207–423) differ in conformation of ring A and orientation of the ligand molecule in the VDR pocket. 1α,25‐Dihydroxyvitamin D3 possessing the A‐ring conformation with axially oriented 1α‐hydroxy group binds receptor with its 25‐hydroxy substituent oriented toward the center of the receptor cavity, whereas ligand possessing equatorial conformation of 1α‐hydroxy enters the pocket with A ring directed inward. The latter conformation and orientation of the ligand is consistent with the crystal structure of hVDR deletion mutant (118–425, Δ [165–215]). The lattice model of rVDR (120–422, Δ [164–207]) shows excellent agreement with the crystal structure of the hVDR mutant. The complex obtained from docking the hormone into the receptor has lower energy than complexes for which homology modeling was used. Thus, a simple model of vitamin D receptor with the first two helices deleted can be potentially useful for designing a general structure of ligand, whereas the advanced lattice model is suitable for examining binding sites in the pocket. Proteins 2001;44:188–199.