Structure, function, dynamics and informatics of macromolecular complexes

Large molecular ‘machines’ comprising multiple protein and/or nucleic acid subunits perform complex, specialized functions in the cell. Research in my laboratory is directed towards clarifying the structural and thermodynamic basis of protein-protein and protein-DNA interactions that regulate the assembly and/or recruitment of these molecular machines. Current efforts are focused on structural and functional characterizations of binary complexes using biochemical and biophysical (primarily, solution state NMR) approaches. These studies address issues relating to the sequence and structural requirements for complex formation, including molecular determinants of affinity, cooperativity, and specificity.

Molecular mechanisms of recruitment and assembly of the Sin3 corepressor complex in transcriptional repression
A major research project that has been ongoing for the past few years involves a comprehensive characterization of interactions mediated by the Sin3 corepressor. Sin3 is a key scaffolding protein of a large (~2 MDa), evolutionarily conserved, multi-protein complex that is required for normal growth and development of tissues and organisms ranging from yeast to human. Sin3 physically associates with multiple components of the complex including chromatin-modifying activities such as histone deacetylases (HDACs), whose actions are crucial for influencing chromatin structure and effecting transcriptional repression, and a surprisingly large number of seemingly unrelated and structurally diverse transcription factors. Members of the Sin3 complex lack intrinsic DNA-binding activity, but can be recruited to specific regions of the genome by sequence-specific DNA binding transcription factors to effect gene silencing. Current studies are aimed at elucidating the molecular mechanisms by which transcription factors recruit Sin3. In the long-term, we will explore aspects relating to the assembly of the corepressor complex itself focusing in particular on interactions involving Sin3 and HDACs, since they appear to be mediated by multiple components of the complex.

Molecular mechanisms of ubiquitin recognition in endocytosis
Another major project in the lab is a characterization of molecular interactions involving monoubiquitin. Ubiquitination is a well-characterized signal for proteosome-mediated degradation of cellular proteins. Recent studies have revealed additional cellular roles for conjugated ubiquitin in endosomal sorting, gene regulation, intra-nuclear localization, and budding of retroviral virions. The ubiquitin signals appear to be transmitted through direct physical interactions with a variety of ubiquitin-binding motifs found in proteins that participate in the aforementioned processes. For example, at least five distinct motifs including the CUE (similar to a domain in the yeast Cue1 protein), UBA (ubiquitin-associated), UIM (ubiquitin interacting motif), UEV (ubiquitin E2 variant), and VHS (Vps27/Hrs/STAM) motifs have been implicated in efficient and accurate targeting of ubiquitinated membrane proteins to the destination organelle during endocytosis. Current studies are focused on how the ubiquitin signal is recognized by these motifs at the molecular level. Future studies will explore the molecular basis of cooperativity involving possibly higher-order associations with component(s) of the endocytic machinery and the role of ubiquitin-binding motifs in transcription regulation.

Methods for automated analysis of macromolecular interactions
Since macromolecular complexes are an important focus of our research, we are developing a web application that detects stabilizing intermolecular interactions in macromolecular complexes from atomic coordinate data. The core software called MONSTER comprises a PERL wrapper that takes advantage of established software in the public domain to validate atomic coordinate files, identify interacting residues, and assign the nature of these interactions. The results are integrated and presented in an intuitive and interactive graphical format. Immediate applications of MONSTER range from mining and validating experimentally-determined structures to guiding mutational analysis. Future extensions include automated sequence motif identification and development of a high-throughput version for creating and mining a relational database. A beta test version of the software is available at http://monster.northwestern.edu.

K. Brubaker, S. M. Cowley, K. Huang, L. Loo, G. S. Yochum, D. E. Ayer, R. N. Eisenman, and I. Radhakrishnan (2000). Solution structure of the interacting domains of the Mad-Sin3 complex: Implications for recruitment of a chromatin-modifying repression complex. Cell 103, 655-665.

R. S. Kang, C. M. Daniels, S. A. Francis, S. C. Shih, W. J. Salerno, L. Hicke, and I. Radhakrishnan (2003). Solution structure of a CUE-ubiquitin complex reveals a conserved mode of ubiquitin binding. Cell 113, 621-630.

Salerno, W.J., Seaver, S.M., Armstrong, B.R., and Radhakrishnan, I. (2004). Monster: Inferring non-covalent interactions in macromolecular structures from atomic coordinate data. Nucleic Acids Res. 32, W566-W568.

Swanson, K.A., Knoepfler, P.S., Huang, K., Kang, R.S., Cowley, S.M., Laherty, C.D., Eisenman, R.N., and Radhakrishnan, I. (2004). HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations. Nat. Struct. Mol. Biol. 11(8): 738-46.

K. A. Swanson, R. S. Kang, S. D. Stamenova, L. Hicke, and I. Radhakrishnan (2003). Solution structure of Vps27 UIM-ubiquitin complex important for endosomal sorting and receptor downregulation. EMBO J. 22, 4597-4606.

View all publications by publications by Ishwar Radhakrishnan listed in the National Library of Medicine (PubMed).