Structural and dynamic studies of biological macromolecules (mainly proteins) and their interactions using NMR spectroscopy
Studies of herpesviral proteins.
Herpes simplex virus 1 (HSV-1) causes diseases that range from painful skin lesions to keratitis and encephalitis. HSV-1 encodes an essential protein called ICP27 that is involved in a diversity of functions during viral infection. ICP27 has the intriguing ability to interact with viral mRNA and a multiplicity of host cell proteins, hijacking hem to benefit virus production. Thus, ICP27 could conceivably serve as a target for antiviral intervention. The future development of antivirals requires an understanding of how the cooperative assembly of these essential multicomponent complexes occurs and how the assembly is regulated. We will study the role of cooperativity in protein-protein and protein-RNA interactions mediated by ICP27 and characterize the binding interfaces of its complexes to determine how ICP27 is assembled in different complexes during viral infection, with the goal of revealing the molecular mechanism of its function. The structural information will be used to explore the role of interaction interface residues in ICP27 function during infection. ICP27 consists of a number of structured as well as intrinsically-unstructured domains, which participate in a large number of diverse protein-protein and protein-RNA interactions. We hypothesize that these interactions may be multi-site and therefore cooperative, mediated by regions that may be distant in the primary sequence. We postulate that transient interactions also are possible, especially when unfolded regions are involved. We hypothesize that at least some of ICP27's interactions are regulated by phosphorylation and/or arginine methylation. We will test these hypotheses in two specific aims: 1) To study the direct binding interfaces of ICP27 with cellular proteins and viral mRNA by generating shorter protein constructs in which structural stability is not perturbed, and expressing them in amounts sufficient for structural biology studie including traditional and novel Isotopically-Discriminated (IDIS) NMR spectroscopy as well as biophysical approaches. Cooperativity will be analyzed in interactions between short functional fragments of intrinsically unstructured N-terminal ICP27 and partners that interact within this region, including viral RNA and host cell proteins. In vitro post-translational modifications of ICP27 will be performed to explore how ICP27-multi-protein complex assembly is affected by phosphorylation and arginine methylation. We will also endeavor to perform structural studies of folded domains from the C-terminal part of ICP27 and to characterize interactions with its cellular protein partners. Because ICP27 undergoes a head-to-tail intramolecular interaction in vivo, a chimera containing appropriate fragments of the N- and C-termini could be created to study how these regions interact. 2) To explore the role of interaction interface residues in ICP27 function during viral infection, recombinant viruses will be constructed bearing point mutations at interaction interfaces and will be characterized for in vivo interactions and ICP27 functional activities and effects on viral infection.
Public Health Relevance
Herpes simplex virus 1 (HSV-1) causes diseases that range from recurrent painful skin lesions to blindness resulting from keratitis and morbidity and mortality due to encephalitis. Recent studies suggest that HSV-1 is a contributing factor in Alzheimer's disease. Further, HSV infection increases the risk for HIV acquisition. Current antivirals do not prevent or fully suppress HSV replication, which raises a major health concern. All human herpesviruses encode an essential multifunctional protein, which is called ICP27 in HSV. ICP27 has the intriguing ability to be involved in a diversity of functions during infection, involving hijacking host macromolecular complexes to benefit virus production. ICP27 interacts with viral mRNA and a multiplicity of host cell proteins and thus could be a target for antiviral intervention. How the cooperative assembly of ICP27 in these complexes occurs and how it is regulated, is unclear, but is required for future development of targeted antivirals directed at interaction interfaces. Further, what we learn about ICP27 may be helpful in designing drug targets for other herpesviruses, such as KSHV, EBV and HCMV, which encode ICP27 homologues.
Development of NMR methods for monitoring complex multiple protein-protein-ligand(s) interactions
Modern physico-chemical and biochemical methods allow thorough characterisation of protein-protein interactions, by measuring some kind of "reporter signal" reflecting these interactions, or by capturing a snapshot - 3D structure of complex. But what if the interaction involves three or more components, which exhibit consecutive, cooperative or concurrent binding: how can we tell what happens dynamically with each existing component as new components are added? Limited number of reporter signals no longer can adequately represent such a complexity. Monitoring such dynamic and transitional events which all are elements of protein interaction networks is intrinsically difficult. Our current research is aimed at development of new isotopically discriminated (IDIS) NMR approaches enabling to monitor complex multi-component binding events in vitro (Golovanov et al., J. Amer. Chem. Soc 2007, 129(20):6528). We have recently also started developing methods how to monitor behaviour of pharmaceutically-relevant proteins in solution.
Reducing aggregation and self-association of pharmaceutically-relevant proteins
Increasing protein concentration in solution to the required level without causing aggregation and precipitation is often a challenging but important task in many fields, including of structural biology, and NMR spectroscopy. Recently the importance of this was further highlighted with the ever increasing number of proteins (such as monoclonal antibodies, mAbs) being approved as drugs by regulatory bodies. Increasing mAb formulation stability for long-term storage, and reducing its viscosity for injections, are the major tasks that pharmaceutical industry now faces. Earlier in our work we discovered that a simple addition of 50 mM L-Arginine and L-Glutamate to the sample buffer significantly increases protein solubility and suppresses proteolytic degradation. The solubilisation protocol has been successfully used for a number of proteins with solubility problems, suggesting that it may be universally applicable. Recently, we demonstrated that arginine glutamate is also effective in increasing stability of mAb formulations.
Protein structure determination
NMR is a powerful method allowing determination of atomic-resolution structures of proteins. Some proteins fail to crystallize due to the presence of flexible tails or loops, and thus are not amenable for X-ray analysis: NMR is the method of choice in these cases. We use a combination of X-ray crystallography and NMR analysis to analyse comprehensively the structure and dynamics of proteins and cooperativity of their interactions with their targets – other proteins, DNA and RNA.