- The Chapman group applies biophysical and other approaches to viral-host interactions, and to understanding enzyme mechanism and dynamics. X-ray diffraction is a common denominator, but, with the help of collaborators, electron microscopy (EM) and NMR allow us to tackle large and dynamic bio-molecular complexes. Our goals are mechanism of action at a molecular level, so structurally directed probes of function, through molecular virology or enzyme kinetics, figure just as prominently. The group is drawn to challenges beyond standard technologies, so we develop the computational approaches for hybrid methodology, by which disparate data can be integrated into a holistic understanding.
Structural Virology - Host Interactions:
Treatments for genetic diseases are just now emerging as a practical reality. In 2012, Glybera™, a treatment for lipoprotein lipase deficiency, became the first gene therapy approved for clinical use in Europe while Nathwani and colleagues report exciting results from clinical trials of a treatment for hemophilia B. In these and other cases, the transgene is delivered to target cells, in vivo, packaged inside an AAV capsid, the protein shell that encloses the single-stranded DNA of this non-pathogenic human parvovirus.
To improve the prospects for treating a broader array of inherited diseases, improvements are needed in the efficiency and specificity with which therapeutic DNA can be delivered to afflicted cells. Interaction of viral surface proteins with cellular receptors and immune molecules are central to understanding the lifecycles of pathogenic viruses. For gene therapy, our understanding of such interactions is put to the test in attempts to evade immune neutralization, and to ellicit efficient and specific cellular targeting.
Our atomic stucture of Adeno-Associated Virus (AAV-2), determined by X-ray crystallography in 2002 (right) laid one of the foundations in developing AAV as a gene therapy vector. Our subsequent structures of the most common human variants, AAV-3 & AAV-6, together with an EM structure of an AAV-2 complex with a neutralizing antibody, have provided insights into how AAV naturally evolves to escape immune neutralization. Our EM structure of AAV-DJ, a recombinant variant developed to target liver cells, showed that it was a major antigenic epitope that had been selected for change, highlighting the role of immune interactions in tissue tropism.
Our current main focus is on the interactions of cell entry. Using both surface plasmon resonance and EM, even now at resolutions beyond 3 Å, we have shown that glycan binding is less specific than one assumed, and that its role is not that of a classical receptor, but in initial adherence to the cell surface. Failing to get evidence of physical interactions with any of several proteins implicated as co-receptors, we have collaborated with Jan Carette (Stanford) on a screening for genes essential to cell entry. This identified an uncharacterized host protein, that we named AAVR, onto which AAV binds, hitching a ride on its natural recycling route to the perinuclear trans Golgi network. AAVR's identification breaks a 20-year impasse, opening the way to a molecular understanding of viral entry.
The term "Protein Structure" is widely used, but a misleading over-simplification. Databases of static structures mis-represent the dynamic nature of proteins which is crucial to function but so difficult to characterize. Our model system for characterizing functional dynamics is arginine kinase, one of the enzymes that buffers cellular
ATP levels. It is a two-substrate enzyme that is larger than previous paradigms, and therefore exemplifies a more representative repertoire of domain rotations and loop motions.
Our crystal structures show a large conformational change on substrate-binding. NMR
relaxation dispersion and residual dipolar coupling have indicated an equilibrium in the substrate-free form, reflecting intrinsic dynamics in the hinged domain and loop re-orientations seen crystallographically. Transition state analog crystal structures at ambient temperature show some directional correlation between thermal displacements and substrate-associated conformational changes, even though only 5% of the magnitude, indicating that "induced" conformational changes take advantage of preferred modes of motion that occur intrinsically. NMR-derived exchange rates revealed that some of the intrinsic motions in substrate-free enzyme are of timescales commensurate with catalytic rate constants. The temperature dependence of both enzyme kinetics and NMR dynamics implicate a common activation barrier, indicating that these protein conformatonal changes are limiting enzyme turnover and that conformational change is by conformational selection. We are currently exploring the dynamics of the transition state form as the next step in a spatial-temporal characterization of functional dynamics through the reaction cycle of an enzyme.
Computational Methods of Refinement
The structures of large biomolecular assemblies are
increasingly studied through electron microscopy (EM) of the whole
complex, combined with detailed crystallography of components. We are developing the computational methods for optimally
fitting component structures into EM maps, through which a more detailed analysis is possible than from EM
alone. The figure shows an early collaboration with Joachim Frank, in which our methods supported detailed analysis of ribosome conformational changes during peptide synthesis at ~ 1nm resolution. As our electron microscopy of AAV has progressed, we have had opportunities to apply the new methods in flexible fitting of atomic models into the EM reconstruction. A challenge on which we are currently focused is the flexible fitting of structures without the over-fitting that occurs with detailed atomic modeling at medium (3 to 6 Å) resolution. Details are on our software page.
Much of the research is interdisciplinary. Group
members are often co-mentored by co-PIs/colleagues experts in other techniques: