The mitotic regulator Survivin binds as a monomer to its functional interactor Borealin

Survivin is a member of the IAP (inhibitor of apoptosis) protein family, defined in part by the presence of a zinc-binding baculoviral inhibitory repeat (BIR) domain. Most BIR domains bind short sequences beginning with alanine, and in this manner, they recognize and block the action of key targets in apoptotic pathways. However, Survivin binds only very weakly to typical IAP ligands. Unique features of Survivin are the long C-terminal helix following the BIR domain and a short segment (linking the helix and BIR domains) that mediates Survivin homodimerization. Despite this detailed knowledge of the structure of Survivin itself, there is a current lack of understanding about how Survivin recognizes cellular binding partners, and consequently, many questions about Survivin function remain unanswered. We determined two co-crystal structures of Survivin and a minimal binding fragment from the chromosomal passenger protein Borealin, a well validated functional interactor. The interaction between Survivin and Borealin involves extensive packing between the long C-terminal helix of Survivin and a long Borealin helix. Surprisingly, an additional important interaction occurs between the Survivin homodimerization interface and a short segment of Borealin. This segment both structurally mimics and displaces one Survivin monomer. The relevance of this unexpected interaction was tested by mutagenesis of two key Borealin residues. Mutant Borealin introduced into HeLa cells failed to localize properly during mitosis and also caused mislocalization of other chromosomal passenger proteins. This suggests that the mutant is dominant-negative and confirms the functional importance of the interaction surface identified in the crystal structures.     

Solution structure of human survivin and its binding interface with Smac/Diablo

NMR studies of the antiapoptotic protein survivin have been used to determine the homodimer interface of the protein in solution and to identify residues of the protein that interact with Smac/Diablo. In solution, survivin(1-120) forms a bow-tie-shaped dimer whose interface is composed of its N-terminal residues as well as residues connecting its BIR domain to the C-terminal alpha helix. The solution structure resolves the controversy regarding the two possible dimer interfaces for survivin observed in X-ray crystal structures. The structural basis for the interaction between survivin and Smac/Diablo was also investigated. When Smac/Diablo or N-terminal Smac/Diablo peptide analogues are added to a solution of survivin, specific residues near alpha4 and beta3 are perturbed. NMR experiments indicate that the peptides bind across the third beta-strand of survivin in a manner similar to the way Smac/Diablo peptides bind to the BIR3 domain of X-linked IAP (XIAP).     

Crystal structure of human survivin reveals a bow tie-shaped dimer with two unusual alpha-helical extensions

Survivin is a mitotic spindle-associated protein involved in linking mitotic spindle function to activation of apoptosis in mammalian cells. The structure of the full-length human survivin has been determined by X-ray crystallography to 2.7 A. Strikingly, the structure forms a very unusual bow tie-shaped dimer. It does not dimerize through a C-terminal coiled-coil, contrary to sequence analysis prediction. The C-terminal helices contain hydrophobic clusters with the potential for protein-protein interactions. The unusual shape and dimensions of survivin suggest it serves an adaptor function through its alpha-helical extensions.     

Discovery of a novel small molecule binding site of human survivin

Survivin is one of the most tumor-specific genes in the human genome and is an attractive target for cancer therapy. However, small-molecule ligands for survivin have not yet been described. Thus, an interrogation of survivin which could potentially both validate a small-molecule therapy approach, and determine the biochemical nature of any of survivin's functions has not been possible. Here we describe the discovery and characterization of a small molecule binding site on the survivin surface distinct from the Smac peptide-binding site. The new site is located at the dimer interface and exhibits many of the features of highly druggable, biologically relevant protein binding sites. A variety of small hydrophobic compounds were found that bind with moderate affinity to this binding site, from which one lead was developed into a group of compounds with nanomolar affinity. Additionally, a subset of these compounds are adequately water-soluble and cell-permeable. Thus, the structural studies and small molecules described here provide tools that can be used to probe the biochemical role(s) of survivin, and may ultimately serve as a basis for the development of small molecule therapeutics acting via direct or allosteric disruption of binding events related to this poorly understood target.     

Survivin study: What is the next wave?

Survivin, a novel member of inhibitor of apoptosis (IAP) protein family, is aberrantly expressed in cancer but undetectable in normal, differentiated adult tissues. Current studies suggest that survivin is implicated in both control of apoptosis and regulation of cell division. However, due to some inconsistent observations on survivin subcellular localization, there is debate about survivin's function in regulating apoptosis, cell division, or both. This review will discuss concepts, experimental methods, and interesting results that unify the different notions about survivin localization and function or point out gaps of knowledge about controversial issues. The author also intends to review various aspects of survivin studies, which were not emphasized or sufficiently discussed in previous reviews on survivin, and update recent developments that may reveal new applications of disease-oriented therapeutics in the coming years.     

Structural Basis for Bivalent Smac-Mimetics Recognition in the IAP Protein Family

XIAP is an apoptotic regulator protein that binds to the effector caspases -3 and -7 through its BIR2 domain, and to initiator caspase-9 through its BIR3 domain. Molecular docking studies suggested that Smac-DIABLO may antagonize XIAP by concurrently targeting both BIR2 and BIR3 domains; on this basis bivalent Smac-mimetic compounds have been proposed and characterized. Here, we report the X-ray crystal structure of XIAP-BIR3 domain in complex with a two-headed compound (compound 3) with improved efficacy relative to its monomeric form. A small-angle X-ray scattering study of XIAP-BIR2BIR3, together with fluorescence polarization binding assays and compound 3 cytotoxicity tests on HL60 leukemia cell line are also reported. The crystal structure analysis reveals a network of interactions supporting XIAP-BIR3/compound 3 recognition; moreover, analytical gel-filtration chromatography shows that compound 3 forms a 1:1 stoichiometric complex with a XIAP protein construct containing both BIR2 and BIR3 domains. On the basis of the crystal structure and small-angle X-ray scattering, a model of the same BIR2-BIR3 construct bound to compound 3 is proposed, shedding light on the ability of compound 3 to relieve XIAP inhibitory effects on caspase-9 as well as caspases -3 and -7. A molecular modeling/docking analysis of compound 3 bound to cIAP1-BIR3 domain is presented, considering that Smac-mimetics have been shown to kill tumor cells by inducing cIAP1 and cIAP2 ubiquitination and degradation. Taken together, the results reported here provide a rationale for further development of compound 3 as a lead in the design of dimeric Smac mimetics for cancer treatment.     

Withanone as an inhibitor of survivin: A potential drug candidate for cancer therapy

Survivin, the smallest inhibitor of apoptosis protein, which has been reported to be highly expressed in almost all known cancers, plays a dual role in survival as well as the proliferation of cancer cells. It inhibits apoptosis by inhibiting caspases as well as facilitating mitosis by becoming a part of chromosomal passenger complex through its BIR5 domain. Docking studies carried out with herbal ligand withanone derived from roots of Withania somnifera have shown strong binding affinity of −19.1088 kJ/mol with BIR5 domain of survivin and in turn interferes with inhibitory action against caspases and may lead to apoptosis. Binding of withanone at BIR5 domain of survivin may also interfere with chromosomal passenger complex and lead to halt the mitotic process within the cancer cell. Docking studies support various experimental outcomes and suggest withanone as a potential candidate molecule in cancer therapy.     

A detailed molecular belt model for apolipoprotein A-I in discoidal high density lipoprotein.

Apolipoprotein A-I (apoA-I) is the principal protein of high density lipoprotein particles (HDL). ApoA-I contains a globular N-terminal domain (residues 1-43) and a lipid-binding C-terminal domain (residues 44-243). Here we propose a detailed model for the smallest discoidal HDL, consisting of two apoA-I molecules wrapped beltwise around a small patch of bilayer containing 160 lipid molecules. The C-terminal domain of each monomer is ringlike, a curved, planar amphipathic alpha helix with an average of 3.67 residues per turn, and with the hydrophobic surface curved toward the lipids. We have explored all possible geometries for forming the dimer of stacked rings, subject to the hypothesis that the optimal geometry will maximize intermolecular salt bridge interactions. The resulting model is an antiparallel arrangement with an alignment matching that of the (nonplanar) crystal structure of lipid-free apoA-I.     

Uncoupling the central spindle-associated function of the chromosomal passenger complex from its role at centromeres

Survivin is a component of the chromosomal passenger complex (CPC) that plays a role in maintenance of an active spindle checkpoint and in cytokinesis. To study whether these different functions can be attributed to distinct domains within the Survivin protein, we complemented Survivin-depleted cells with a variety of point- and deletion-mutants of Survivin. We show that an intact baculovirus IAP repeat (BIR) domain is required for proper spindle checkpoint functioning, but dispensable for cytokinesis. In line with this, mutants lacking an intact BIR domain localized normally to the central spindle, but their localization to inner centromeres was severely perturbed. Consequently, these mutants failed to recruit Aurora B, Borealin/Dasra B, and BubR1 to centromeres and kinetochores, but they had retained the ability to recruit Aurora B and Borealin/Dasra B to the midzone and midbody. Thus, the C terminus of Survivin is sufficient for central spindle localization and execution of cytokinesis, but the additional presence of a functional BIR domain is essential for centromere targeting and spindle checkpoint function. Importantly, our data show that the function of the CPC at the centromere can be separated from its function at the central spindle and that execution of cytokinesis does not require prior concentration of the CPC at centromeres.     

Human survivin is a kinetochore-associated passenger protein

Survivin, a dimeric baculovirus inhibitor of apoptosis repeat (BIR) motif protein that is principally expressed in G2 and mitosis, has been associated with protection against apoptosis of cells that exit mitosis aberrantly. Mammalian survivin has been reported to associate with centrosomes and with the mitotic spindle. We have expressed a human hemagglutinin-tagged survivin plasmid to determine its localization, and find instead that it clearly acts as a passenger protein. In HeLa cells, survivin first associates with the kinetochores, and then translocates to the spindle midzone during anaphase and, finally, to the midbody during cell cleavage. Its localization is similar to that of TD-60, a known passenger protein. Both a point mutation in the baculovirus IAP repeat motif (C84A) and a COOH-terminal deletion mutant (Delta106) of survivin fail to localize to either kinetochores or midbodies, but neither interferes with cell cleavage. The interphase localization of survivin is cell cycle regulated since in permanently transfected NIH3T3 cells it is excluded from the nuclei until G2, where it localizes with centromeres. Survivin remains associated with mitotic kinetochores when microtubule assembly is disrupted and its localization is thus independent of microtubules. We conclude that human survivin is positioned to have an important function in the mechanism of cell cleavage.     

Role of survivin in mitosis

Human survivin is a member of the IAP (Inhibitor of Apoptosis) family. It was reported that survivin expression is associated with drug resistance, cancer progression and low patient survival rate in many cancers. Survivin is implicated in both: inhibition of apoptosis and regulation of cell division. As a member of Chromosomal Passenger Complex (CPC) it is involved in sister chromatids segregation during mitosis. On the other hand, survivin plays an important role in the surveillance mechanism called mitotic spindle assembly checkpoint (MSAC) which regulates metaphase to anaphase transition during mitosis. Additionally, survivin is necessary for cytokinesis progression. The present review is a summary of survivin's functions, focused on its role in cell division in normal and cancer cells, as well as introduction to discussion about anticancer therapies based on survivin depletion.     

An unconventional IAP-binding motif revealed by target-assisted iterative screening (TAIS) of the BIR3-cIAP1 domain

Target-assisted iterative screening (TAIS) has been applied to a random phage-displayed peptide library in a search for novel ligands of the third baculovirus IAP ('inhibitors of apoptosis') repeat (BIR) domain of cIAP1. The peptides selected in the screen fall into two distinct specificity groups, one that conforms to a known IAP-binding motif (IBM) and another one that reveals a novel BIR domain interacting motif, NH(2)-SR(V/P)W. The biochemical profiling of selected sequences with synthetic peptides, which included alanine scanning and N- and C-terminal truncations as well as competition with the Smac peptide, suggests a major energetic contribution of tryptophan at the +4 position of peptide ligands to binding and identifies the latter together with the respective pocket on the BIR domain surface as a 'hot spot' of the interaction. A peptide featuring the novel motif selectively binds the full-length cIAP1 protein in cell lysates. A 'two-pocket' model of BIR domain recognition mechanism is proposed as the basis of differential BIR domain interactions with different IBMs.     

Crystal structure of the M1 protein-binding domain of the influenza A virus nuclear export protein (NEP/NS2)

During influenza virus infection, viral ribonucleoproteins (vRNPs) are replicated in the nucleus and must be exported to the cytoplasm before assembling into mature viral particles. Nuclear export is mediated by the cellular protein Crm1 and putatively by the viral protein NEP/NS2. Proteolytic cleavage of NEP defines an N-terminal domain which mediates RanGTP-dependent binding to Crm1 and a C-terminal domain which binds to the viral matrix protein M1. The 2.6 A crystal structure of the C-terminal domain reveals an amphipathic helical hairpin which dimerizes as a four-helix bundle. The NEP-M1 interaction involves two critical epitopes: an exposed tryptophan (Trp78) surrounded by a cluster of glutamate residues on NEP, and the basic nuclear localization signal (NLS) of M1. Implications for vRNP export are discussed.     

Crystal structure of proteolytic fragments of the redox-sensitive Hsp33 with constitutive chaperone activity

Heat shock protein 33 (Hsp33) inhibits aggregation of partially denatured proteins during oxidative stress. The chaperone activity of Hsp33 is unique among heat shock proteins because the activity is reversibly regulated by cellular redox status. We report here the crystal structure of the N-terminal region of Hsp33 fragments with constitutive chaperone activity. The structure reveals that the N-terminal portion of Hsp33 forms a tightly associated dimer formed by a domain crossover. A concave groove on the dimeric surface contains an elongated hydrophobic patch that could potentially bind denatured protein substrates. The termini of the subunits are located near the hydrophobic patch, indicating that the cleaved C-terminal domain may shield the hydrophobic patch in an inactive state. Two of the four conserved zinc-coordinating cysteines are in the end of the N-terminal domain, and the other two are in the cleaved C-terminal domain. The structural information and subsequent biochemical characterizations suggest that the redox switch of Hsp33 occurs by a reversible dissociation of the C-terminal regulatory domain through oxidation of zinc-coordinating cysteines and zinc release.     

Molecular cloning and characterization of a novel inhibitor of apoptosis protein from Xenopus laevis

A novel inhibitor of apoptosis protein family member termed SIX was identified in Xenopus containing a single baculoviral IAP repeat (BIR) domain and no COOH-terminal RING finger domain. It exhibited striking amino acid sequence similarity with human survivin, mouse TIAP, and recently found Xenopus survivin, especially a part of BIR domain was highly conserved. Interestingly, SIX interacted with RXRalpha through the AF2 domain in the absence of ligand, which was weakened when the ligand was present. Northern blot analysis demonstrated that SIX mRNA was not detectable in adult with exception of the ovary and testis, and whole-mount in situ hybridization and Northern blot analyses revealed strong and homogeneous expression of SIX in the developing oocytes. In the embryos, the expression of SIX was observed in the animal hemisphere from one-cell to yolk plug stages and high level of expression was detected in the future brain and dorsal region of the neural tube at the neurula stage and early tail-bud stage. These results strongly support the fact that survivin is evolutionarily conserved in structure and SIX is likely to be the Xenopus counterpart of human and mouse survivin.     

Mechanism of endophilin N-BAR domain-mediated membrane curvature

Endophilin-A1 is a BAR domain-containing protein enriched at synapses and is implicated in synaptic vesicle endocytosis. It binds to dynamin and synaptojanin via a C-terminal SH3 domain. We examine the mechanism by which the BAR domain and an N-terminal amphipathic helix, which folds upon membrane binding, work as a functional unit (the N-BAR domain) to promote dimerisation and membrane curvature generation. By electron paramagnetic resonance spectroscopy, we show that this amphipathic helix is peripherally bound in the plane of the membrane, with the midpoint of insertion aligned with the phosphate level of headgroups. This places the helix in an optimal position to effect membrane curvature generation. We solved the crystal structure of rat endophilin-A1 BAR domain and examined a distinctive insert protruding from the membrane interaction face. This insert is predicted to form an additional amphipathic helix and is important for curvature generation. Its presence defines an endophilin/nadrin subclass of BAR domains. We propose that N-BAR domains function as low-affinity dimers regulating binding partner recruitment to areas of high membrane curvature.