Book of Abstracts: Albany 2009

category image Albany 2009
Conversation 16
June 16-20 2009
© Adenine Press (2008)

Helix Docking Dynamics: Interface Plasticity and Conformational Dynamics Contributions to Molecular Recognition within a Ubiquitin/UIM Fusion Protein

Ubiquitin-Interacting motifs (UIMs) are an important class of protein domains that interact with proteins of the Ubiquitin fold superfamily (1). These 20 residue-long domains are found in a variety of proteins and serve as modular elements that recognize their intracellular targets, which may be individual ubiquitin subunits, polyubiquitin chains or other ubiquitylated proteins of the cell (2). Previous structural studies of the interactions between UIMs with ubiquitin have shown that UIMs adopt an α-helical structure, which exposes a pattern of hydrophobic residues with a conserved sequence pattern that interacts with key hydrophobic residues on ubiquitin (3, 4). In order to better understand the interactions between UIMs and ubiquitin, as well as the contribution of protein dynamics to domain-domain interactions, we have designed a fusion protein between yeast ubiquitin and a UIM derived from the Vps27p protein. The solution structure of the fusion protein is characterized in atomic resolution and is found to consist of a well-defined ubiquitin core and an amphipathic UIM helix with a flexible C-terminus. The UIM helix docks on a hydrophobic surface defined by the β3 and β4 strands of ubiquitin, and forms additional stabilizing interactions with residues in two turns connecting strands β1 to β2 and strand β3 to the short strand β'. Moreover, we characterize the plasticity of the docking interface, as well as global changes in the microsecond to millisecond protein motions with a variety of NMR relaxation experiments (5). Analysis of our CPMG results (6, 7) suggests the presence of two types of global motions, one directly linked to fluctuations within the UIM interface, the other being induced in distal parts of the molecule as a result of reorganization of intermolecular interactions. These motions affect sites that are directly involved in the interaction of ubiquitin with ubiquitinating enzymes and/or other components of the endocytosis machinery (8), thus may provide a basis for the modulation of these interactions. Molecular Dynamics simulations at the μsec timescale starting from the NMR ensemble suggest a structural model for these correlated motions. This study demonstrates a case where localized interactions among protein domains have long-range effects in protein motions that occur on a variety of timescales for this model ubiquitin-UIM interaction system.

Nikolaos G. Sgourakis1
Scott A. McCallum1
Mayank M. Patel1
Angel E. Garcia2
and George I. Makhatadze1

1Dept. of Biology
2Department of Physics, Applied Physics and Astronomy
Center for Biotechnology and Interdisciplinary Studies
Rensselaer Polytechnic Institute
Troy, NY 12180

Tel: (518) 276-4417
Fax: (518) 276-2851

Backbone dynamics of ubiquitin decrease within the UIM interaction surface upon binding: Changes in backbone chemical shifts and fast timescale motions are shown. Left: sites that undergo chemical shift perturbations closely correlate with those that show a decrease in the generalized order parameter. The importance of the AGQL loop and I44 is particularly notable. Right: changes in the generalized order parameter for backbone N-H groups. Colors are based on groups of ppm distances and order parameter changes respectively, as indicated by the color bars.

References and Footnotes
  1. Hicke, L., Schubert, H. L., & Hill, C. P. Nature reviews 6, 610-621 (2005).
  2. Pickart, C. M. & Fushman, D. Current opinion in chemical biology 8, 610-616 (2004).
  3. Fisher, R. D., Wang, B., Alam, S. L., Higginson, D. S., Robinson, H., Sundquist, W. I., & Hill, C.P.The Journal of biological chemistry 278, 28976-28984 (2003).
  4. Swanson, K. A., Kang, R. S., Stamenova, S. D., Hicke, L., & Radhakrishnan, I. The EMBO journal 22, 4597-4606 (2003).
  5. Palmer, A. G., 3rd Chemical reviews 104, 3623-3640 (2004).
  6. Carr, H. Y. & Purcell, E. M. Physical Review 94, 630-638 (1954).
  7. Carver, J. P. & Richards, R. E. Journal of magnetic resonance 6, 89 (1972).
  8. Bonifacino, J. S. & Traub, L. M. Annual Review of Biochemistry 72, 395-447 (2003).