In silico

Funded by UEFISCDI with the contract 181/10.10.2011

OVERALL PRESENTATION

From recognition processes to DNA processing, proteins are found within cells in transitory or permanent association with other molecules, as complexes or macromolecular assemblies [1]. Unveiling the subunit architecture and dynamics of modular proteins or the structure-function relation in supramolecular assemblies became thus one of the most important tasks of structural biology today [2]. The architecture of modular proteins and of higher order structures built from proteins and/or nucleic acids are experimentally probed with a vast number of techniques such as X-ray crystallography, NMR spectroscopy, electron microscopy or solution scattering [3]. However these are very tedious and often subjected to failure and limitations, in the first two cases; or of low resolution, in the rest of the cases.

Alternatively much effort was lately devoted to develop less expensive and faster computational techniques such as docking and use them to model assemblage [4]. Despite progresses many challenges still lies ahead in this field too, due to the poorness of empirical forcefields resulting in a reduced ability to discriminate between large numbers of alternative solutions [5].

Nevertheless, when used in combination with constraints derrived from string and structural bioinformatics, and/or with data from experimental techniques such as FRET or mass spectrometry the power of computational techniques in investigating assemblies and assemblage greatly improves. The present project aims to explore more closely this possibility by developing strategies and tools for assisting in-silico the experimental work related to the investigation of molecular complexes and assemblies, and test them in as many problems as possible in structural biology.

Among other applications, particular attention will be devoted to using the techniques developed herein to three topics: -a) the investigation of protein-protein interactions responsible for the architecture of resistance proteins in plants and their recognition of pathogen effectors proteins that trigger plant immune response; -b) the investigation of protein-protein and protein-DNA interactions explaining the difference in functioning of human topoisomerases and the effects of some point mutations proven to induce differential malignant effects in topo II α & β; and -c) The investigation of Paired Complex architecture with focuss on the DNA configuration within the complex, and of the protein-DNA and protein-protein interactions in this transient molecular assembly.

Attention to protein-protein interactions responsible for the architecture of resistance proteins in plants and their recognition of pathogen effector proteins that trigger plant immune response is of high economic relevance. Our Department has actively participated to the advances in this field during the European integrated project FP6-BIOEXPLOIT (FOOD-CT-2005-513959) in which we coordinated the workpackage WP2.4 dedicated to the probabilistic structural modeling of new genomic sequences identified as being involved in these interactions. Within the frame of the present project we intend to tacke this work a step further and complement the collaborative activities provided by the COST action FA1208 "EFECTOME: Pathogen-informed strategies for sustainable broad-spectrum crop resistance" which aims: "to provide a platform for a strong European network to coordinate research on the molecular bases and evolution of effectors-mediated pathogenicity, with the goal of developing unified strategies for durable, broad-range resistance".

The interest in understanding the structural basis of the difference in functioning of the two human topoisomerases is mainly linked to their different response to drugs and radiation in cancer treatment. Our interest in topoII enzymes was raised by a collaboration with a team from Levine Cancer Institute, North Carolina headed by Professor Ram Ganapathi with which we previously located by molecular modelling the position of important point mutations in monomeric topoisomerases. Remaining questions remain to pinpoint these mutations within the functional dimers and assess if these are involved in the internal dynamics of the machine or in the recruitment of other proteins forming the assemblage that ultimately is responsible for the overall DNA processing. Additional questions are related not only to the location and effect of PTM in the two isozymes but to the more general aspects related to the distribution of point mutations on the surface and protein core, and how these do affect the plugging of the two molecular machines in the cellular context, responsible for the divergence of topo II α & β functional phenotypes.

The third major topics raised from the collaboration of DBSB with the Laboratory of David Schatz, from the Department of Immunobiology of Yale School of Medicine on some structural biology aspects of RAG-DNA complexes. In conjunction with molecular modeling and simulation FRET and smFRET data will be used to investigate structural relationships within this assembly. The research on RAG-DNA complexes is sustained in the partner lab by the NIH grant 4R37 AI032524-21 (2012-2017) in which the project leader of the present application is involved as collaborator. In this context, the present application is aimed to complement the NIH grant with the work related to developing experimentally constrained modeling techniques in RAG-DNA complexes.

References
1. Ban, Engelman, "Struct of Large Cellular Assemblies", Curr.Op.Str.Biol, 20, 207-209 (2010)
2. Rey, Sundquist, "Macromolecular Assemblage", Curr.Op.Str.Biol, 21, 221-222 (2011)
3. Rey, Saibil, "Macromolecular Assemblies", Curr.Op.Str.Biol, 19, 178-180 (2009)
4. Stein et al, "3D modeling of protein interactions", Curr.Op.Str.Biol, 21, 200-208 (2011)
5. Vajda, Kozakov, "Convergence in prot-prot docking", Curr.Op.Str.Biol, 19, 164-170 (2009)