UC San Diego

Most complex viruses undergo a capsid maturation process from a precursor, or procapsid particle form to a more stable, infectious particle. dsDNA bacteriophage in particular, exhibit enormous conformational changes in their capsids, as hundreds of protein subunits must undergo a coordinated rearrangement that enables the particle to package and withstand high pressures of DNA. The reorganization in these phage often includes expansion of the capsid diameter, and significant thinning of the capsid shell, facilitated by subunit rotations. In vitro maturation experiments have shown that for many systems, the process can be mimicked without DNA packaging, instead using heat or perturbation agents to stimulate maturation, characterized as a highly exothermic event. Unlike most motor proteins such as ATP synthase, myosin, and many others, the process is uncoupled to hydrolysis of NTP's, instead using a trapped assembly intermediate as the driving force for the process. The structural mechanism for this behavior has been unknown for systems that undergo large-scale rearrangements such as dsDNA bacteriophage. We have been able to crystallize the pre- expanded particle form of bacteriophage HK97 at near atomic resolution and have been able to compare it to a previously solved structure of the mature HK97 capsid, enabling a first look at the structural mechanism associated with HK97 capsid maturation. The study has enabled us to identify the Prohead II state as an intermediate folded state that undergoes refolding and tertiary twisting motions to accommodate particle maturation, in contrast to the previously posited theories implicating rigid body movements as the method of transition. We have also used H/2H exchange coupled to MALDI mass spectrometry to follow the refolding transitions during expansion, as subunits hinge around newly identified 3-fold interactions that remain fixed throughout expansion, stabilizing inter-capsomer contacts and leveraging the transition. The study provides insight into a mechanism of expansion that consists of local high energy conformations maintained by quaternary interactions in the procapsid state, that when perturbed to expand, undergo an irreversible refolding transition that stabilizes the particle