Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. In this study, we describe a novel cell lysis mechanism for fungal and tumor cells by the plant defensin NaD1 that acts via direct binding to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). We determined the crystal structure of a NaD1:PIP2 complex, revealing a striking oligomeric arrangement comprising seven dimers of NaD1 that cooperatively bind the anionic headgroups of 14 PIP2 molecules through a unique ‘cationic grip’ configuration. Site-directed mutagenesis of NaD1 confirms that PIP2-mediated oligomerization is important for fungal and tumor cell permeabilization. These observations identify an innate recognition system by NaD1 for direct binding of PIP2 that permeabilizes cells via a novel membrane disrupting mechanism.DOI: http://dx.doi.org/10.7554/eLife.01808.001

Apoptosis is an important part of the host's defense mechanism for eliminating invading pathogens. Some viruses express proteins homologous in sequence and function to mammalian pro-survival Bcl-2 proteins. Anti-apoptotic F1L expressed by vaccinia virus is essential for survival of infected cells, but it bears no discernable sequence homology to proteins other than its immediate orthologues in related pox viruses. Here we report that the crystal structure of F1L reveals a Bcl-2-like fold with an unusual N-terminal extension. The protein forms a novel domain-swapped dimer in which the a1 helix is the exchanged domain. Binding studies reveal an atypical BH3-binding profile, with sub-micromolar affinity only for the BH3 peptide of pro-apoptotic Bim and low micromolar affinity for the BH3 peptides of Bak and Bax. This binding interaction is sensitive to F1L mutations within the predicted canonical BH3-binding groove, suggesting parallels between how vaccinia virus F1L and myxoma virus M11L bind BH3 domains. Structural comparison of F1L with other Bcl-2 family members reveals a novel sequence signature that redefines the BH4 domain as a structural motif present in both pro-and anti-apoptotic Bcl-2 members, including viral Bcl-2-like proteins. In higher organisms, programmed cell death (apoptosis) is a prominent feature of the response to viral infection. The central role of the Bcl-2 protein family in maintaining cell survival or driving apoptosis, thereby removing infected, damaged or unwanted cells, is reflected by the expression of sequence, structural and functional orthologues of Bcl-2 by certain viruses. 1 The Bcl-2-related proteins share the presence of one or more of four Bcl-2 homology (BH) domains in their primary sequences and act either to promote cell survival or to counter this. 2 Pro-survival family members such as mammalian Bcl-2, Bcl-x L , Bcl-w, Mcl-1 and A1 block apoptosis until their protective effect is countered by binding of proapoptotic BH3-only proteins, such as Bim, Bad or Noxa. 2,3 Pro-survival Bcl-2 proteins contain multiple BH domains, whereas the distantly related BH3-only proteins contain only the a-helical BH3 domain, which binds a receptor-like groove on the pro-survival proteins, thereby inactivating them. 4,5 Upon activation, pro-apoptotic Bax and Bak, which are essential for apoptosis to proceed, 6 oligomerize to cause organellar damage.The viral Bcl-2-like proteins, including those expressed by adenovirus, Kaposi sarcoma-associated herpesvirus, Epstein-Barr virus (EBV) and g-herpesvirus 68, are all required for successful viral propagation and/or persistence. However, other viruses express anti-apoptotic proteins that are unrelated by sequence to any known cell death regulator. These include the myxoma virus M11L, 7 cytomegalovirus vMIA 8 and vaccinia virus F1L 9 and E3L. 10 Despite the lack of sequence similarity, M11L adopts a Bcl-2-like fold. 11 Moreover, the structure of M11L in complex with the BH3 peptide from Bak revealed that the canonical BH3-binding groove is utilized in...

Thrombospondins (TSPs) are extracellular regulators of cell-matrix interactions and cell phenotype. The most highly conserved region of all TSPs are the calcium-binding type 3 (T3) repeats and the C-terminal globular domain (CTD). The crystal structure of a cell-binding TSP-1 fragment, spanning three T3 repeats and the CTD, reveals a compact assembly. The T3 repeats lack secondary structure and are organised around a core of calcium ions; two DxDxDGxxDxxD motifs per repeat each encapsulate two calcium ions in a novel arrangement. The CTD forms a lectin-like b-sandwich and contains four strictly conserved calcium-binding sites. Disruption of the hairpin structure of T3 repeats 6 and 7 decreases protein secretion and stability. The availability for cell attachment of an RGD motif in T3 repeat 7 is modulated by calcium loading. The central architectural role of calcium explains how it is critical for the functions of the TSP C-terminal region. Mutations in the T3 repeats of TSP-5/COMP, which cause two human skeletal disorders, are predicted to disrupt the tertiary structure of the T3-CTD assembly.

Many viruses express antiapoptotic proteins to counter host defense mechanisms that would otherwise trigger the rapid clearance of infected cells. For example, adenoviruses and some gamma-herpesviruses express homologs of prosurvival Bcl-2 to subvert the host's apoptotic machinery. Myxoma virus, a double-stranded DNA virus of the pox family, harbors antiapoptotic M11L, its virulence factor. Analysis of its three-dimensional structure reveals that despite lacking any primary sequence similarity to Bcl-2, it adopts a virtually identical protein fold. This allows it to associate with BH3 domains, especially those of Bax and Bak. We found that M11L acts primarily by sequestering Bax and Bak, thereby blocking the killing action of these essential cell-death mediators. These findings expand the family of protein sequences that act like Bcl-2 to block apoptosis and support the conclusion that the prosurvival action of these proteins critically depends on their ability to bind and antagonize Bax and/or Bak.

Epstein-Barr virus (EBV) is associated with human malignancies, especially those affecting the B cell compartment such as Burkitt lymphoma. The virally encoded homolog of the mammalian pro-survival protein Bcl-2, BHRF1 contributes to viral infectivity and lymphomagenesis. In addition to the pro-apoptotic BH3-only protein Bim, its key target in lymphoid cells, BHRF1 also binds a selective sub-set of pro-apoptotic proteins (Bid, Puma, Bak) expressed by host cells. A consequence of BHRF1 expression is marked resistance to a range of cytotoxic agents and in particular, we show that its expression renders a mouse model of Burkitt lymphoma untreatable. As current small organic antagonists of Bcl-2 do not target BHRF1, the structures of it in complex with Bim or Bak shown here will be useful to guide efforts to target BHRF1 in EBV-associated malignancies, which are usually associated with poor clinical outcomes.

SummaryThe discoidin domain receptors, DDR1 and DDR2, are widely expressed receptor tyrosine kinases that are activated by triple-helical collagen. They control important aspects of cell behavior and are dysregulated in several human diseases. The major DDR2-binding site in collagens I–III is a GVMGFO motif (O is hydroxyproline) that also binds the matricellular protein SPARC. We have determined the crystal structure of the discoidin domain of human DDR2 bound to a triple-helical collagen peptide. The GVMGFO motifs of two collagen chains are recognized by an amphiphilic pocket delimited by a functionally critical tryptophan residue and a buried salt bridge. Collagen binding results in structural changes of DDR2 surface loops that may be linked to the process of receptor activation. A comparison of the GVMGFO-binding sites of DDR2 and SPARC reveals a striking case of convergent evolution in collagen recognition.

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Two phylogenetically and structurally distinct groups of proteins regulate stress induced intrinsic apoptosis, the programmed disassembly of cells. Together they form the B cell lymphoma-2 (Bcl-2) family. Bcl-2 proteins appeared early in metazoan evolution and are identified by the presence of up to four short conserved sequence blocks known as Bcl-2 homology (BH) motifs, or domains. The simple BH3-only proteins bear only a BH3-motif and are intrinsically disordered proteins and antagonize or activate the other group, the multi-motif Bcl-2 proteins that have up to four BH motifs, BH1-BH4. Multi-motif Bcl-2 proteins are either pro-survival or pro-apoptotic in action and have remarkably similar α-helical bundle structures that provide a binding groove formed from the BH1, BH2, and BH3-motifs for their BH3-bearing antagonists. In mammals a network of interactions between Bcl-2 members regulates mitochondrial outer membrane permeability (MOMP) and efflux of cytochrome c and other death inducing factors from mitochondria to initiate the apoptotic caspase cascade, but the molecular events leading to MOMP are uncertain. Dysregulation of the Bcl-2 family occurs in many diseases and pathogenic viruses have assimilated pro-survival Bcl-2 proteins to evade immune responses. Their role in disease has made the Bcl-2 family the focus of drug design attempts and clinical trials are showing promise for 'BH3-mimics', drugs that mimic the ability of BH3-only proteins to neutralize selected pro-survival proteins to induce cell death in tumor cells. This review focuses on the structural biology of Bcl-2 family proteins, their interactions and attempts to harness them as targets for drug design.