Anne Pettikiriarachchi

Membrane Remodeling by the Double-Barrel Scaffolding Protein of Poxvirus

In contrast to most enveloped viruses, poxviruses produce infectious particles that do not acquire their internal lipid membrane by budding through cellular compartments. Instead, poxvirus immature particles are generated from atypical crescent-shaped precursors whose architecture and composition remain contentious. Here we describe the 2.6 Å crystal structure of vaccinia virus D13, a key structural component of the outer scaffold of viral crescents. D13 folds into two jellyrolls decorated by a head domain of novel fold. It assembles into trimers that are homologous to the double-barrel capsid proteins of adenovirus and lipid-containing icosahedral viruses. We show that, when tethered onto artificial membranes, D13 forms a honeycomb lattice and assembly products structurally similar to the viral crescents and immature particles. The architecture of the D13 honeycomb lattice and the lipid-remodeling abilities of D13 support a model of assembly that exhibits similarities with the giant mimivirus. Overall, these findings establish that the first committed step of poxvirus morphogenesis utilizes an ancestral lipid-remodeling strategy common to icosahedral DNA viruses infecting all kingdoms of life. Furthermore, D13 is the target of rifampicin and its structure will aid the development of poxvirus assembly inhibitors.

Crystal structure of a BCL-W domain-swapped dimer: implications for the function of BCL-2 family proteins.

The prosurvival and proapoptotic proteins of the BCL-2 family share a similar three-dimensional fold despite their opposing functions. However, many biochemical studies highlight the requirement for conformational changes for the functioning of both types of proteins, although structural data to support such changes remain elusive. Here, we describe the X-ray structure of dimeric BCL-W that reveals a major conformational change involving helices α3 and α4 hinging away from the core of the protein. Biochemical and functional studies reveal that the α4-α5 hinge region is required for dimerization of BCL-W, and functioning of both pro- and antiapoptotic BCL-2 proteins. Hence, this structure reveals a conformational flexibility not seen in previous BCL-2 protein structures and provides insights into how these regulators of apoptosis can change conformation to exert their function.

The Functional Differences between Pro-survival and Pro-apoptotic B Cell Lymphoma 2 (Bcl-2) Proteins Depend on Structural Differences in Their Bcl-2 Homology 3 (BH3) Domains

Background: Anti- and pro-apoptotic B cell lymphoma 2 (Bcl-2) proteins possess Bcl-2 homology 3 (BH3) domains generally associated with cell death induction. Results: Features of anti-apoptotic BH3 domains were identified that limit their killing activity but are important for protein stability. Conclusion: Pro- and anti-apoptotic BH3 domains are distinct, which affects their function. Significance: Differences in BH3 domains have significant consequences in apoptotic signaling. Bcl-2 homology 3 (BH3) domains are short sequence motifs that mediate nearly all protein-protein interactions between B cell lymphoma 2 (Bcl-2) family proteins in the intrinsic apoptotic cell death pathway. These sequences are found on both pro-survival and pro-apoptotic members, although their primary function is believed to be associated with induction of cell death. Here, we identify critical features of the BH3 domains of pro-survival proteins that distinguish them functionally from their pro-apoptotic counterparts. Biochemical and x-ray crystallographic studies demonstrate that these differences reduce the capacity of most pro-survival proteins to form high affinity “BH3-in-groove” complexes that are critical for cell death induction. Switching these residues for the corresponding residues in Bcl-2 homologous antagonist/killer (Bak) increases the binding affinity of isolated BH3 domains for pro-survival proteins; however, their exchange in the context of the parental protein causes rapid proteasomal degradation due to protein destabilization. This is supported by further x-ray crystallographic studies that capture elements of this destabilization in one pro-survival protein, Bcl-w. In pro-apoptotic Bak, we demonstrate that the corresponding distinguishing residues are important for its cell-killing capacity and antagonism by pro-survival proteins.

Physiological restraint of Bak by Bcl-xL is essential for cell survival

Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-xL Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-xL via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-xL in apoptotic regulation. In vitro, Bak(Q75L) cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-xL binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak.

Ultramarine, a Chromoprotein Acceptor for Forster Resonance Energy Transfer

We have engineered a monomeric blue non-fluorescent chromoprotein called Ultramarine (fluorescence quantum yield, 0.001; ε 585 nm, 64,000 M−1. cm−1) for use as a Förster resonance energy transfer acceptor for a number of different donor fluorescent proteins. We show its use for monitoring activation of caspase 3 in live cells using fluorescence lifetime imaging. Ultramarine has the potential to increase the number of cellular parameters that can be imaged simultaneously.

The BECN1 N-terminal domain is intrinsically disordered

ABSTRACT BECN1/Beclin 1 has a critical role in the early stages of autophagosome formation. Recently, structures of its central and C-terminal domains were reported, however, little structural information is available on the N-terminal domain, comprising a third of the protein. This lack of structural information largely stems from the inability to produce this region in a purified form. Here, we describe the expression and purification of the N-terminal domain of BECN1 (residues 1 to 150) and detailed biophysical characterization, including NMR spectroscopy. Combined, our studies demonstrated at the atomic level that the BECN1 N-terminal domain is intrinsically disordered, and apart from the BH3 subdomain, remains disordered following interaction with a binding partner, BCL2L1/BCL-XL. In addition, the BH3 domain α-helix induced upon interaction with BCL2L1 reverts to a disordered state when the complex is dissociated by exposure to a competitive inhibitor. No significant interactions between N- and C-terminal domains were detected.

Characterisation of the conformational preference and dynamics of the intrinsically disordered N-terminal region of Beclin 1 by NMR spectroscopy

Beclin 1 is a 450 amino acid protein that plays critical roles in the early stages of autophagosome formation. We recently reported the successful expression, purification and structural characterisation of the entire N-terminal region of Beclin 1 (residues 1-150), including its backbone NMR chemical shift assignments. Based on assigned backbone NMR chemical shifts, it has been established that the N-terminal region of Beclin 1 (1-150), including the BH3 domain (112-123), is intrinsically disordered in the absence of its interaction partners. Here, a detailed study of its conformational preference and backbone dynamics obtained from an analysis of its secondary structure populations using the δ2D method, and the measurements of effective hydrodynamic radius as well as (1)H temperature coefficients, (1)H solvent exchange rates, and (15)N relaxation parameters of backbone amides using NMR spectroscopy is reported. These data provide further evidence for the intrinsically disordered nature of the N-terminal region of Beclin 1 and support the view that the helical conformation adopted by the Beclin 1 BH3 domain upon interaction with binding partners such as BCL-2 pro-survival proteins is likely induced rather than pre-existing.

A transgenic mouse model to inducibly target prosurvival Bcl2 proteins with selective BH3 peptides in vivo.

A transgenic mouse model to inducibly target prosurvival Bcl2 proteins with selective BH3 peptides in vivo