Jijie Chai

A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis.

X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspase-9 and inhibits its activity, whereas Smac (also known as DIABLO) relieves this inhibition through interaction with XIAP. Here we show that XIAP associates with the active caspase-9–Apaf-1 holoenzyme complex through binding to the amino terminus of the linker peptide on the small subunit of caspase-9, which becomes exposed after proteolytic processing of procaspase-9 at Asp 315. Supporting this observation, point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9, or deletion of the linker peptide, prevented caspase-9 association with XIAP and its concomitant inhibition. We note that the N-terminal four residues of caspase-9 linker peptide share significant homology with the N-terminal tetra-peptide in mature Smac and in the Drosophila proteins Hid/Grim/Reaper, defining a conserved class of IAP-binding motifs. Consistent with this finding, binding of the caspase-9 linker peptide and Smac to the BIR3 domain of XIAP is mutually exclusive, suggesting that Smac potentiates caspase-9 activity by disrupting the interaction of the linker peptide of caspase-9 with BIR3. Our studies reveal a mechanism in which binding to the BIR3 domain by two conserved peptides, one from Smac and the other one from caspase-9, has opposing effects on caspase activity and apoptosis.

Structural and biochemical basis of apoptotic activation by Smac/DIABLO.

Apoptosis (programmed cell death), an essential process in the development and homeostasis of metazoans, is carried out by caspases. The mitochondrial protein Smac/DIABLO performs a critical function in apoptosis by eliminating the inhibitory effect of IAPs (inhibitor of apoptosis proteins) on caspases. Here we show that Smac/DIABLO promotes not only the proteolytic activation of procaspase-3 but also the enzymatic activity of mature caspase-3, both of which depend upon its ability to interact physically with IAPs. The crystal structure of Smac/DIABLO at 2.2 Å resolution reveals that it homodimerizes through an extensive hydrophobic interface. Missense mutations inactivating this dimeric interface significantly compromise the function of Smac/DIABLO. As in the Drosophila proteins Reaper, Grim and Hid, the amino-terminal amino acids of Smac/DIABLO are indispensable for its function, and a seven-residue peptide derived from the amino terminus promotes procaspase-3 activation in vitro. These results establish an evolutionarily conserved structural and biochemical basis for the activation of apoptosis by Smac/DIABLO.

Structural basis of IAP recognition by Smac/DIABLO.

Apoptosis is an essential process in the development and homeostasis of all metazoans. The inhibitor-of-apoptosis (IAP) proteins suppress cell death by inhibiting the activity of caspases; this inhibition is performed by the zinc-binding BIR domains of the IAP proteins. The mitochondrial protein Smac/DIABLO promotes apoptosis by eliminating the inhibitory effect of IAPs through physical interactions. Amino-terminal sequences in Smac/DIABLO are required for this function, as mutation of the very first amino acid leads to loss of interaction with IAPs and concomitant loss of Smac/DIABLO function. Here we report the high-resolution crystal structure of Smac/DIABLO complexed with the third BIR domain (BIR3) of XIAP. Our results show that the N-terminal four residues (Ala-Val-Pro-Ile) in Smac/DIABLO recognize a surface groove on BIR3, with the first residue Ala binding a hydrophobic pocket and making five hydrogen bonds to neighbouring residues on BIR3. These observations provide a structural explanation for the roles of the Smac N terminus as well as the conserved N-terminal sequences in the Drosophila proteins Hid/Grim/Reaper. In conjunction with other observations, our results reveal how Smac may relieve IAP inhibition of caspase-9 activity. In addition to explaining a number of biological observations, our structural analysis identifies potential targets for drug screening.

Mechanism of XIAP-Mediated Inhibition of Caspase-9.

The inhibitor of apoptosis (IAP) proteins potently inhibit the catalytic activity of caspases. While profound insight into the inhibition of the effector caspases has been gained in recent years, the mechanism of how the initiator caspase-9 is regulated by IAPs remains enigmatic. This paper reports the crystal structure of caspase-9 in an inhibitory complex with the third baculoviral IAP repeat (BIR3) of XIAP at 2.4 A resolution. The structure reveals that the BIR3 domain forms a heterodimer with a caspase-9 monomer. Strikingly, the surface of caspase-9 that interacts with BIR3 also mediates its homodimerization. We demonstrate that monomeric caspase-9 is catalytically inactive due to the absence of a supporting sequence element that could be provided by homodimerization. Thus, XIAP sequesters caspase-9 in a monomeric state, which serves to prevent catalytic activity. These studies, in conjunction with other observations, define a unified mechanism for the activation of all caspases.

Structural Basis of Caspase-7 Inhibition by XIAP

The inhibitor of apoptosis (IAP) proteins suppress cell death by inhibiting the catalytic activity of caspases. Here we present the crystal structure of caspase-7 in complex with a potent inhibitory fragment from XIAP at 2.45 A resolution. An 18-residue XIAP peptide binds the catalytic groove of caspase-7, making extensive contacts to the residues that are essential for its catalytic activity. Strikingly, despite a reversal of relative orientation, a subset of interactions between caspase-7 and XIAP closely resemble those between caspase-7 and its tetrapeptide inhibitor DEVD-CHO. Our biochemical and structural analyses reveal that the BIR domains are dispensable for the inhibition of caspase-3 and -7. This study provides a structural basis for the design of the next-generation caspase inhibitors.

Pseudomonas syringae Effector AvrPto Blocks Innate Immunity by Targeting Receptor Kinases

Plants use receptor kinases, such as FLS2 and EFR, to perceive bacterial pathogens and initiate innate immunity. This immunity is often suppressed by bacterial effectors, allowing pathogen propagation. To counteract, plants have evolved disease resistance genes that detect the bacterial effectors and reinstate resistance. The Pseudomonas syringae effector AvrPto promotes infection in susceptible plants but triggers resistance in plants carrying the protein kinase Pto and the associated resistance protein Prf. Here we show that AvrPto binds receptor kinases, including Arabidopsis FLS2 and EFR and tomato LeFLS2, to block plant immune responses in the plant cell. The ability to target receptor kinases is required for the virulence function of AvrPto in plants. The FLS2-AvrPto interaction and Pto-AvrPto interaction appear to share similar sequence requirements, and Pto competes with FLS2 for AvrPto binding. The results suggest that the mechanism by which AvrPto recognizes virulence targets is linked to the evolution of Pto, which, in association with Prf, recognizes the bacterium and triggers strong resistance.

Structural basis for flg22-induced activation of the Arabidopsis FLS2-BAK1 immune complex.

First Defense In defense against bacterial infection, plants carry a cell-surface receptor, known as FLS2, that can bind to a fragment of bacterial flagellin and trigger defense responses. Y. Sun et al. (p. 624, published online 10 October) investigated the structural details that govern the binding between FLS2, its co-receptor BAK1, and the flagellin fragment flg22. The assembled complex initiates signals to activate the plants innate immune response. The molecular basis for how a plant heterodimeric receptor responds to bacterial infection signals is elucidated. Flagellin perception in Arabidopsis is through recognition of its highly conserved N-terminal epitope (flg22) by flagellin-sensitive 2 (FLS2). Flg22 binding induces FLS2 heteromerization with BRASSINOSTEROID INSENSITIVE 1–associated kinase 1 (BAK1) and their reciprocal activation followed by plant immunity. Here, we report the crystal structure of FLS2 and BAK1 ectodomains complexed with flg22 at 3.06 angstroms. A conserved and a nonconserved site from the inner surface of the FLS2 solenoid recognize the C- and N-terminal segment of flg22, respectively, without oligomerization or conformational changes in the FLS2 ectodomain. Besides directly interacting with FLS2, BAK1 acts as a co-receptor by recognizing the C terminus of the FLS2-bound flg22. Our data reveal the molecular mechanisms underlying FLS2-BAK1 complex recognition of flg22 and provide insight into the immune receptor complex activation.

Chitin-Induced Dimerization Activates a Plant Immune Receptor

Dissecting Chitin Binding The chitin in fungal cells walls serves as a trigger to initiate plant defenses against pathogenic fungi. Arabidopsis detects these signals through a cell surface chitin receptor whose intracellular kinase domain initiates a signaling cascade in response to chitin that activates the plants response to infection. Liu et al. (p. 1160) have now solved the crystal structure of the Arabidopsis chitin receptor AtCERK1. The results show how chitin binds to the receptor and suggest that the biological response requires dimerisation of the receptor when it binds a chitin oligomer at least seven or eight subunits long. Structural analysis shows how fungus-derived chitin dimerizes its receptor on target plants and triggers defense responses. Pattern recognition receptors confer plant resistance to pathogen infection by recognizing the conserved pathogen-associated molecular patterns. The cell surface receptor chitin elicitor receptor kinase 1 of Arabidopsis (AtCERK1) directly binds chitin through its lysine motif (LysM)–containing ectodomain (AtCERK1-ECD) to activate immune responses. The crystal structure that we solved of an AtCERK1-ECD complexed with a chitin pentamer reveals that their interaction is primarily mediated by a LysM and three chitin residues. By acting as a bivalent ligand, a chitin octamer induces AtCERK1-ECD dimerization that is inhibited by shorter chitin oligomers. A mutation attenuating chitin-induced AtCERK1-ECD dimerization or formation of nonproductive AtCERK1 dimer by overexpression of AtCERK1-ECD compromises AtCERK1-mediated signaling in plant cells. Together, our data support the notion that chitin-induced AtCERK1 dimerization is critical for its activation.

Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi.

HtrA2/Omi, a mitochondrial serine protease in mammals, is important in programmed cell death. However, the underlining mechanism of HtrA2/Omi-mediated apoptosis remains unclear. Analogous to the bacterial homolog HtrA (DegP), the mature HtrA2 protein contains a central serine protease domain and a C-terminal PDZ domain. The 2.0 Å crystal structure of HtrA2/Omi reveals the formation of a pyramid-shaped homotrimer mediated exclusively by the serine protease domains. The peptide-binding pocket of the PDZ domain is buried in the intimate interface between the PDZ and the protease domains. Mutational analysis reveals that the monomeric HtrA2/Omi mutants are unable to induce cell death and are deficient in protease activity. The PDZ domain modulates HtrA2/Omi-mediated cell death activity by regulating its serine protease activity. These structural and biochemical observations provide an important framework for deciphering the mechanisms of HtrA2/Omi-mediated apoptosis.

Crystal Structure of a Phosphorylated Smad2: Recognition of Phosphoserine by the MH2 Domain and Insights on Smad Function in TGF-β Signaling

Ligand-induced phosphorylation of the receptor-regulated Smads (R-Smads) is essential in the receptor Ser/Thr kinase-mediated TGF-beta signaling. The crystal structure of a phosphorylated Smad2, at 1.8 A resolution, reveals the formation of a homotrimer mediated by the C-terminal phosphoserine (pSer) residues. The pSer binding surface on the MH2 domain, frequently targeted for inactivation in cancers, is highly conserved among the Co- and R-Smads. This finding, together with mutagenesis data, pinpoints a functional interface between Smad2 and Smad4. In addition, the pSer binding surface on the MH2 domain coincides with the surface on R-Smads that is required for docking interactions with the serine-phosphorylated receptor kinases. These observations define a bifunctional role for the MH2 domain as a pSer-X-pSer binding module in receptor Ser/Thr kinase signaling pathways.