Peter E. Prevelige

Preliminary crystallographic analysis of the bacteriophage P22 portal protein

Portal proteins are components of large oligomeric dsDNA pumps connecting the icosahedral capsid of tailed bacteriophages to the tail. Prior to the tail attachment, dsDNA is actively pumped through a central cavity formed by the subunits. We have studied the portal protein of bacteriophage P22, which is the largest connector characterized among the tailed bacteriophages. The molecular weight of the monomer is 82.7 kDa, and it spontaneously assembles into an oligomeric structure of approximately 1.0 MDa. Here we present a preliminary biochemical and crystallographic characterization of this large macromolecular complex. The main difficulties related to the crystallization of P22 portal protein lay in the intrinsic dynamic nature of the portal oligomer. Recombinant connectors assembled from portal monomers expressed in Escherichia coli form rings of different stoichiometry in solution, which cannot be separated on the basis of their size. To overcome this intrinsic heterogeneity we devised a biochemical purification that separates different ring populations on the basis of their charge. Small ordered crystals were grown from drops containing a high concentration of the kosmotropic agent tert-butanol and used for data collection. A preliminary crystallographic analysis to 7.0-A resolution revealed that the P22 portal protein crystallized in space group I4 with unit cell dimensions a=b=409.4A, c=260.4A. This unit cell contains a total of eight connectors. Analysis of the noncrystallographic symmetry by the self-rotation function unambiguously confirmed that bacteriophage P22 portal protein is a dodecamer with a periodicity of 30 degrees. The cryo-EM reconstruction of the dodecahedral bacteriophage T3 portal protein will be used as a model to initiate phase extension and structure determination.

A retroviral chimeric capsid protein reveals the role of the N-terminal β-hairpin in mature core assembly.

The human immunodeficiency virus (HIV) is an enveloped virus constituted by two monomeric RNA molecules that encode for 15 proteins. Among these are the structural proteins that are translated as the gag polyprotein. In order to become infectious, HIV must undergo a maturation process mediated by the proteolytic cleavage of gag to give rise to the isolated structural protein matrix, capsid (CA), nucleocapsid as well as p6 and spacer peptides 1 and 2. Upon maturation, the 13 N-terminal residues from CA fold into a β-hairpin, which is stabilized mainly by a salt bridge between Pro1 and Asp51. Previous reports have shown that non-formation of the salt bridge, which potentially disrupts proper β-hairpin arrangement, generates noninfectious virus or aberrant cores. To date, however, there is no consensus on the role of the β-hairpin. In order to shed light in this subject, we have generated mutations in the hairpin region to examine what features would be crucial for the β-hairpins role in retroviral mature core formation. These features include the importance of the proline at the N-terminus, the amino acid sequence, and the physical structure of the β-hairpin itself. The presented experiments provide biochemical evidence that β-hairpin formation plays an important role in regard to CA protein conformation required to support proper mature core arrangement. Hydrogen/deuterium exchange and in vitro assembly reactions illustrated the importance of the β-hairpin structure, its dynamics, and its influence on the orientation of helix 1 for the assembly of the mature CA lattice.

In vitro incorporation of the phage Phi29 connector complex.

The incorporation of the DNA packaging connector complex during lambdoid phage assembly in vivo is strictly controlled-one and only one of the twelve identical icosahedral vertices is differentiated by the inclusion of a portal or connector dodecamer. Proposed control mechanisms include obligate nucleation from a connector containing complex, addition of the connector as the final step during assembly, and a connector-mediated increase in the growth rate. The inability to recapitulate connector incorporation in vitro has made it difficult to obtain direct biochemical evidence in support of one model over another. Here we report the development an in vitro assembly system for the well characterized dsDNA phage Phi29 which results in the co-assembly of connector with capsid and scaffolding proteins to form procapsid-like particles (PLPs). Immuno-electron microscopy demonstrates the specific incorporation of connector vertex in PLPs. The connector protein increases both the yield and the rate of capsid assembly suggesting that the incorporation of the connector in Phi29 likely promotes nucleation of assembly.

Kinetic and mass spectrometry-based investigation of human immunodeficiency virus type 1 assembly and maturation.

Publisher Summary Advances in mass spectrometry have made it possible to ionize proteins and peptides without fragmentation. It holds particular promise for application to the analysis of complex macromolecular machines, where it serves as a complement to the traditional approaches of X-ray crystallography and electron microscopy. This chapter describes the application of mass spectrometry to the analysis of the assembly and maturation of human immunodeficiency virus type 1 (HIV-1). Electron tomography has the potential to deliver reconstructions of HIV1 specimens but the beam damage associated with repeatedly imaging biological specimens currently limits the resolution. The use of a combination of hydrogen–deuterium exchange mass spectrometry and chemical cross-linking provides a pathway to obtain intermediate-resolution information that can be used to identify the inter subunit interfaces and physical arrangement of subunits within complex biological structures.

Structural insights into the stabilization of the human immunodeficiency virus type 1 capsid protein by the cyclophilin-binding domain and implications on the virus cycle.

During infection, human immunodeficiency virus type 1 (HIV-1) interacts with the cellular host factor cyclophilin A (CypA) through residues 85-93 of the N-terminal domain of HIV-1s capsid protein (CA). The role of the CA:CypA interaction is still unclear. Previous studies showed that a CypA-binding loop mutant, Δ87-97, has increased ability to assemble in vitro. We used this mutant to infer whether the CypA-binding region has an overall effect on CA stability, as measured by pressure and chemical perturbation. We built a SAXS-based envelope model for the dimer of both WT and Δ87-97. A new conformational arrangement of the dimers is described, showing the structural plasticity that CA can adopt. In protein folding studies, the deletion of the loop drastically reduces CA stability, as assayed by high hydrostatic pressure and urea. We hypothesize that the deletion promotes a rearrangement of helix 4, which may enhance the heterotypic interaction between the N- and C-terminal domains of CA dimers. In addition, we propose that the cyclophilin-binding loop may modulate capsid assembly during infection, either in the cytoplasm or near the nucleus by binding to the nuclear protein Nup385.

Targeted mutagenesis of the P22 portal protein reveals the mechanism of signal transmission during DNA packaging

The portal vertex in dsDNA bacteriophage serves as the site for genome encapsidation and release. In several of these viruses, efficient termination of DNA packaging has been shown to be dependent on the density of packaged DNA. The portal protein has been implicated as being part of the sensor that regulates packaging termination through DNA-dependent conformational changes during packaging. The mechanism by which DNA induces these conformational changes remains unknown. In this study, we explore how point mutants in the portal core can result in changes in genome packaging density in P22. Mutations in the portal core that subtly alter the structure or dynamics of the protein result in an increase in the amount of DNA packaged. The magnitude of the change is amino acid and location specific. Our findings suggest a mechanism wherein compression of the portal core is an essential aspect of signal transmission during packaging.

ϕ29 Scaffolding and connector structure-function relationship studied by trans-complementation

A dodecamer of connector protein forms a conduit at a unique five-fold vertex in the capsid of many dsDNA-containing viruses providing the means for DNA entry and egress. The molecular mechanism guiding the incorporation of one connector per procapsid remains obscure; however, a recent bacteriophage ϕ29 model suggests that incorporation is coupled to nucleation between the connector and scaffolding proteins and particular amino acids may promote interactions between the two proteins. To test this model in vivo, a trans-complementation system using cloned scaffolding genes was implemented and tested for the ability to complement a ϕ29 amber-scaffolding strain. Wild type scaffolding gene induction resulted in efficient virion production, whereas synthesis of mutant scaffolding proteins displayed various phenotypes. Biochemical analyses of the resultant particles substantiate the previously identified amino acid residues in connector incorporation. Furthermore, kinetic studies of virion production using the in vivo trans-complementation system support the nucleation model.