Structure (v.20, #1)
In This Issue (v-vii).
The Rise of Oxygen and Aerobic Biochemistry by Mak A. Saito (1-2).
Analysis of conserved protein folding domains across extant genomes by Kim et al. in this issue of Structure provides insights into the timing of some of the earliest aerobic metabolisms to arise on Earth.
Couple Dynamics: PPARγ and Its Ligand Partners by Shanghai Yu; H. Eric Xu (2-4).
Ligand-regulated transcriptional activity is the most important property of nuclear receptors, including PPARγ. In this issue of Structure, Hughes et al. determined how the dynamic conformations of ligands and the receptor contribute to the degree of ligand-dependent activation of PPARγ, which provide further insights into design of PPARγ-based anti-diabetic drugs.
Computational Design of Membrane Proteins by Jose Manuel Perez-Aguilar; Jeffery G. Saven (5-14).
Membrane proteins are involved in a wide variety of cellular processes, and are typically part of the first interaction a cell has with extracellular molecules. As a result, these proteins comprise a majority of known drug targets. Membrane proteins are among the most difficult proteins to obtain and characterize, and a structure-based understanding of their properties can be difficult to elucidate. Notwithstanding, the design of membrane proteins can provide stringent tests of our understanding of these crucial biological systems, as well as introduce novel or targeted functionalities. Computational design methods have been particularly helpful in addressing these issues, and this review discusses recent studies that tailor membrane proteins to display specific structures or functions and examines how redesigned membrane proteins are being used to facilitate structural and functional studies.Display Omitted
Conditional Peripheral Membrane Proteins: Facing up to Limited Specificity by Katarina Moravcevic; Camilla L. Oxley; Mark A. Lemmon (15-27).
Regulated relocalization of signaling and trafficking proteins is crucial for the control of many cellular processes and is driven by a series of domains that respond to alterations at membrane surfaces. The first examples of these domains—conditional peripheral membrane proteins—included C1, C2, PH, PX, and FYVE domains, which specifically recognize single tightly regulated membrane components such as diacylglycerol or phosphoinositides. The structural basis for this recognition is now well understood. Efforts to identify additional domains with similar functions that bind other targets (or participate in unexplained cellular processes) have not yielded many more examples of specific phospholipid-binding domains. Instead, most of the recently discovered conditional peripheral membrane proteins bind multiple targets (each with limited specificity), relying on coincidence detection and/or recognizing broader physical properties of the membrane such as charge or curvature. This broader range of recognition modes presents significant methodological challenges for a full structural understanding.
Telomerase and Telomere-Associated Proteins: Structural Insights into Mechanism and Evolution by Karen A. Lewis; Deborah S. Wuttke (28-39).
Recent advances in our structural understanding of telomerase and telomere-associated proteins have contributed significantly to elucidating the molecular mechanisms of telomere maintenance. The structures of telomerase TERT domains have provided valuable insights into how experimentally identified conserved motifs contribute to the telomerase reverse transcriptase reaction. Additionally, structures of telomere-associated proteins in a variety of organisms have revealed that, across evolution, telomere-maintenance mechanisms employ common structural elements. For example, the single-stranded 3′ overhang of telomeric DNA is specifically and tightly bound by an OB-fold in nearly all species, including ciliates (TEBP and Pot1a), fission yeast (SpPot1), budding yeast (Cdc13), and humans (hPOT1). Structures of the yeast Cdc13, Stn1, and Ten1 proteins demonstrated that telomere maintenance is regulated by a complex that bears significant similarity to the RPA heterotrimer. Similarly, proteins that specifically bind double-stranded telomeric DNA in divergent species use homeodomains to execute their functions (human TRF1 and TRF2 and budding yeast ScRap1). Likewise, the conserved protein Rap1, which is found in budding yeast, fission yeast, and humans, contains a structural motif that is known to be critical for protein-protein interaction. In addition to revealing the common underlying themes of telomere maintenance, structures have also elucidated the specific mechanisms by which many of these proteins function, including identifying a telomere-specific domain in Stn1 and how the human TRF proteins avoid heterodimerization. In this review, we summarize the high-resolution structures of telomerase and telomere-associated proteins and discuss the emergent common structural themes among these proteins. We also address how these high-resolution structures complement biochemical and cellular studies to enhance our understanding of telomere maintenance and function.
NMR Structure of a Heterodimeric SAM:SAM Complex: Characterization and Manipulation of EphA2 Binding Reveal New Cellular Functions of SHIP2 by Hyeong J. Lee; Prasanta K. Hota; Preeti Chugha; Hong Guo; Hui Miao; Liqun Zhang; Soon-Jeung Kim; Lukas Stetzik; Bing-Cheng Wang; Matthias Buck (41-55).
The sterile alpha motif (SAM) for protein-protein interactions is encountered in over 200 proteins, but the structural basis for its interactions is just becoming clear. Here we solved the structure of the EphA2-SHIP2 SAM:SAM heterodimeric complex by use of NMR restraints from chemical shift perturbations, NOE and RDC experiments. Specific contacts between the protein surfaces differ significantly from a previous model and other SAM:SAM complexes. Molecular dynamics and docking simulations indicate fluctuations in the complex toward alternate, higher energy conformations. The interface suggests that EphA family members bind to SHIP2 SAM, whereas EphB members may not; correspondingly, we demonstrate binding of EphA1, but not of EphB2, to SHIP2. A variant of EphB2 SAM was designed that binds SHIP2. Functional characterization of a mutant EphA2 compromised in SHIP2 binding reveals two previously unrecognized functions of SHIP2 in suppressing ligand-induced activation of EphA2 and in promoting receptor coordinated chemotactic cell migration.► A multi-ensemble solution structure of a SAM:SAM heterodimer was solved by NMR ► Mutagenesis (residue pair swaps) confirm the most populated ensemble ► Structure is different from a previous model and other SAM:SAM complexes ► Modification of the surface and in vivo study reveals new functions for the complex
Mechanism of Regulation of Receptor Histidine Kinases by Hedda U. Ferris; Stanislaw Dunin-Horkawicz; Nora Hornig; Michael Hulko; Jörg Martin; Joachim E. Schultz; Kornelius Zeth; Andrei N. Lupas; Murray Coles (56-66).
Bacterial transmembrane receptors regulate an intracellular catalytic output in response to extracellular sensory input. To investigate the conformational changes that relay the regulatory signal, we have studied the HAMP domain, a ubiquitous intracellular module connecting input to output domains. HAMP forms a parallel, dimeric, four-helical coiled coil, and rational substitutions in our model domain (Af1503 HAMP) induce a transition in its interhelical packing, characterized by axial rotation of all four helices (the gearbox signaling model). We now illustrate how these conformational changes are propagated to a downstream domain by fusing Af1503 HAMP variants to the DHp domain of EnvZ, a bacterial histidine kinase. Structures of wild-type and mutant constructs are correlated with ligand response in vivo, clearly associating them with distinct signaling states. We propose that altered recognition of the catalytic domain by DHp, rather than a shift in position of the phospho-accepting histidine, forms the basis for regulation of kinase activity.► The HAMP and DHp domains of a histidine kinase communicate via rotational signals ► Rotation peaks at a conserved charged layer at the HAMP-DHp domain junction ► The position of the phospho-accepting histidine is not modulated during signaling ► The enzyme is regulated by specific recognition of the catalytic domain by DHp
Protein Domain Structure Uncovers the Origin of Aerobic Metabolism and the Rise of Planetary Oxygen by Kyung Mo Kim; Tao Qin; Ying-Ying Jiang; Ling-Ling Chen; Min Xiong; Derek Caetano-Anollés; Hong-Yu Zhang; Gustavo Caetano-Anollés (67-76).
The origin and evolution of modern biochemistry remain a mystery despite advances in evolutionary bioinformatics. Here, we use a structural census in nearly 1,000 genomes and a molecular clock of folds to define a timeline of appearance of protein families linked to single-domain enzymes. The timeline sorts out enzymatic recruitment, validates patterns in metabolic history, and reveals that the most ancient reaction of aerobic metabolism involved the synthesis of pyridoxal 5′-phosphate or pyridoxal and appeared 2.9 Gyr ago. The oxygen source for this primordial reaction was probably Mn catalase, which appeared at the same time and could have generated oxygen as a side product of hydrogen peroxide detoxification. Finally, evolutionary analysis of transferred groups and metabolite fragments revealed that oxidized sulfur did not participate in metabolism until the rise of oxygen. The evolutionary patterns we uncover in molecules and chemistries provide strong support for the coevolution of biochemistry and geochemistry.► The primordial reaction involved the synthesis of pyridoxal 5′-phosphate or pyridoxal ► Mn catalase produced oxygen as a side product of hydrogen peroxide detoxification ► Evolution of amino acid biosynthesis and codon-amino acid assignments was congruent ► Biochemistry and geochemistry coevolved
The Alternatively Spliced Acid Box Region Plays a Key Role in FGF Receptor Autoinhibition by Juliya Kalinina; Kaushik Dutta; Dariush Ilghari; Andrew Beenken; Regina Goetz; Anna V. Eliseenkova; David Cowburn; Moosa Mohammadi (77-88).
Uncontrolled fibroblast growth factor (FGF) signaling can lead to human malignancies necessitating multiple layers of self-regulatory control mechanisms. Fibroblast growth factor receptor (FGFR) autoinhibition mediated by the alternatively spliced immunoglobulin (Ig) domain 1 (D1) and the acid box (AB)-containing linker between D1 and Ig domain 2 (D2) serves as the first line of defense to minimize inadvertent FGF signaling. In this report, nuclear magnetic resonance and surface plasmon resonance spectroscopy are used to demonstrate that the AB subregion of FGFR electrostatically engages the heparan sulfate (HS)-binding site on the D2 domain in cis to directly suppress HS-binding affinity of FGFR. Furthermore, the cis electrostatic interaction sterically autoinhibits ligand-binding affinity of FGFR because of the close proximity of HS-binding and primary ligand-binding sites on the D2 domain. These data, together with the strong amino acid sequence conservation of the AB subregion among FGFR orthologs, highlight the universal role of the AB subregion in FGFR autoinhibition.► The AB region interacts intramolecularly with the HBS on the D2 domain of FGFR ► The cis AB:HBS interaction competitively autoinhibits HS-binding affinity of FGFR ► The cis AB:HBS interaction sterically suppresses ligand-binding affinity of FGFR ► The AB-mediated FGFR autoinhibiton is a universal mechanism for FGFR regulation
Structure of the Yersinia pestis FabV Enoyl-ACP Reductase and Its Interaction with Two 2-Pyridone Inhibitors by Maria W. Hirschbeck; Jochen Kuper; Hao Lu; Nina Liu; Carla Neckles; Sonam Shah; Steffen Wagner; Christoph A. Sotriffer; Peter J. Tonge; Caroline Kisker (89-100).
The recently discovered FabV enoyl-ACP reductase, which catalyzes the last step of the bacterial fatty acid biosynthesis (FAS-II) pathway, is a promising but unexploited drug target against the reemerging pathogen Yersinia pestis. The structure of Y. pestis FabV in complex with its cofactor reveals that the enzyme features the common architecture of the short-chain dehydrogenase reductase superfamily, but contains additional structural elements that are mostly folded around the usually flexible substrate-binding loop, thereby stabilizing it in a very tight conformation that seals the active site. The structures of FabV in complex with NADH and two newly developed 2-pyridone inhibitors provide insights for the development of new lead compounds, and suggest a mechanism by which the substrate-binding loop opens to admit the inhibitor, a motion that could also be coupled to the interaction of FabV with the acyl-carrier protein substrate.► Structure of the recently discovered enoyl-ACP reductase FabV (+NADH) of Y.pestis Research Highlight ► Substrate-binding loop stabilized by additional structural elements in FabV Research Highlight ► Structures of two ternary complexes in the presence of 2-pyridone inhibitors Research Highlight ► The ternary structures provide clues to substrate binding
Structure of Ddn, the Deazaflavin-Dependent Nitroreductase from Mycobacterium tuberculosis Involved in Bioreductive Activation of PA-824 by Susan E. Cellitti; Jennifer Shaffer; David H. Jones; Tathagata Mukherjee; Meera Gurumurthy; Badry Bursulaya; Helena I. Boshoff; Inhee Choi; Amit Nayyar; Yong Sok Lee; Joseph Cherian; Pornwaratt Niyomrattanakit; Thomas Dick; Ujjini H. Manjunatha; Clifton E. Barry; Glen Spraggon; Bernhard H. Geierstanger (101-112).
Tuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F420 deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F420 and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data.Display Omitted► Crystal structures of Ddn, the reductase for antitubercular drug PA-824 ► Active site residues and F420-binding mode described ► The N terminus is important for PA-824 binding but could not be fully characterized ► An active homolog from Nocardia farcinica was identified and studied
Computational Reconstruction of Multidomain Proteins Using Atomic Force Microscopy Data by Minh-Hieu Trinh; Michael Odorico; Michael E. Pique; Jean-Marie Teulon; Victoria A. Roberts; Lynn F. Ten Eyck; Elizabeth D. Getzoff; Pierre Parot; Shu-wen W. Chen; Jean-Luc Pellequer (113-120).
Classical structural biology techniques face a great challenge to determine the structure at the atomic level of large and flexible macromolecules. We present a novel methodology that combines high-resolution AFM topographic images with atomic coordinates of proteins to assemble very large macromolecules or particles. Our method uses a two-step protocol: atomic coordinates of individual domains are docked beneath the molecular surface of the large macromolecule, and then each domain is assembled using a combinatorial search. The protocol was validated on three test cases: a simulated system of antibody structures; and two experimentally based test cases: Tobacco mosaic virus, a rod-shaped virus; and Aquaporin Z, a bacterial membrane protein. We have shown that AFM-intermediate resolution topography and partial surface data are useful constraints for building macromolecular assemblies. The protocol is applicable to multicomponent structures connected in the polypeptide chain or as disjoint molecules. The approach effectively increases the resolution of AFM beyond topographical information down to atomic-detail structures.Display Omitted► Combining high-resolution AFM imaging with computational reconstruction protocol ► Method combines AFM surface topography and computational docking ► Aims at reducing the gap in the knowledge of 3D structures of very large protein ► Atomic force microscopy, a new player in integrative structural biology
The Transmembrane Protein KpOmpA Anchoring the Outer Membrane of Klebsiella pneumoniae Unfolds and Refolds in Response to Tensile Load by Patrick D. Bosshart; Iordan Iordanov; Carlos Garzon-Coral; Pascal Demange; Andreas Engel; Alain Milon; Daniel J. Müller (121-127).
In Klebsiella pneumoniae the transmembrane β-barrel forming outer membrane protein KpOmpA mediates adhesion to a wide range of immune effector cells, thereby promoting respiratory tract and urinary infections. As major transmembrane protein OmpA stabilizes Gram-negative bacteria by anchoring their outer membrane to the peptidoglycan layer. Adhesion, osmotic pressure, hydrodynamic flow, and structural deformation apply mechanical stress to the bacterium. This stress can generate tensile load to the peptidoglycan-binding domain (PGBD) of KpOmpA. To investigate how KpOmpA reacts to mechanical stress, we applied a tensile load to the PGBD and observed a detailed unfolding pathway of the transmembrane β-barrel. Each step of the unfolding pathway extended the polypeptide connecting the bacterial outer membrane to the peptidoglycan layer and absorbed mechanical energy. After relieving the tensile load, KpOmpA reversibly refolded back into the membrane. These results suggest that bacteria may reversibly unfold transmembrane proteins in response to mechanical stress.Display Omitted► Exposed to mechanical stress KpOmpA unfolds stepwise transmembrane β-strands ► The last unfolded structural element (N-terminal β-strand) is the strongest anchor in the membrane ► In absence of mechanical stress, KpOmpA refolds into the lipid membrane ► Reversible unfolding of membrane proteins may protect bacteria against mechanical stress
A Master Switch Couples Mg2+-Assisted Catalysis to Domain Motion in B. stearothermophilus Tryptophanyl-tRNA Synthetase by Violetta Weinreb; Li Li; Charles W. Carter (128-138).
We demonstrate how tryptophanyl-tRNA synthetase uses conformation-dependent Mg2+ activation to couple catalysis of tryptophan activation to specific, functional domain movements. Rate acceleration by Mg2+ requires ∼−6.0 kcal/mol in protein⋅Mg2+ interaction energy, none of which arises from the active site. A highly cooperative interaction between Mg2+ and four residues from a remote, conserved motif that mediates the shear of domain movement (1) destabilizes the pretransition state conformation, thereby (2) inducing the Mg2+ to stabilize the transition state for kcat by ∼−5.0 kcal/mol. Cooperative, long-range conformational effects on the metal therefore convert an inactive Mg2+ coordination into one that can stabilize the transition state if, and only if, domain motion occurs. Transient, conformation-dependent Mg2+ activation, analogous to the escapement in mechanical clocks, explains vectorial coupling.Display Omitted► Combinatorial mutagenesis at four remote sites shifts reaction coordinate stabilities ► Cooperative action of all four sites accounts for the entire catalytic role of Mg2+ ► Catalysis of ATP utilization by Mg2+ occurs if, and only if, the conformation changes ► This type of allosteric effect may explain vectorial coupling in transducing NTPases
Ligand and Receptor Dynamics Contribute to the Mechanism of Graded PPARγ Agonism by Travis S. Hughes; Michael J. Chalmers; Scott Novick; Dana S. Kuruvilla; Mi Ra Chang; Theodore M. Kamenecka; Mark Rance; Bruce A. Johnson; Thomas P. Burris; Patrick R. Griffin; Douglas J. Kojetin (139-150).
Ligand binding to proteins is not a static process, but rather involves a number of complex dynamic transitions. A flexible ligand can change conformation upon binding its target. The conformation and dynamics of a protein can change to facilitate ligand binding. The conformation of the ligand, however, is generally presumed to have one primary binding mode, shifting the protein conformational ensemble from one state to another. We report solution nuclear magnetic resonance (NMR) studies that reveal peroxisome proliferator-activated receptor γ (PPARγ) modulators can sample multiple binding modes manifesting in multiple receptor conformations in slow conformational exchange. Our NMR, hydrogen/deuterium exchange and docking studies reveal that ligand-induced receptor stabilization and binding mode occupancy correlate with the graded agonist response of the ligand. Our results suggest that ligand and receptor dynamics affect the graded transcriptional output of PPARγ modulators.Display Omitted► PPARγ ligands can sample multiple binding modes in slow exchange ► Multiple binding modes manifest in distinct receptor conformations in slow exchange ► Graded agonism correlates with differences in receptor dynamics ► Both ligand and receptor dynamics impact function
Structural Basis for Molecular Interactions Involving MRG Domains: Implications in Chromatin Biology by Tao Xie; Richard Graveline; Ganesan Senthil Kumar; Yongbo Zhang; Arvind Krishnan; Gregory David; Ishwar Radhakrishnan (151-160).
MRG15 is a member of the mortality family of transcription factors that targets a wide variety of multiprotein complexes involved in transcription regulation, DNA repair, and alternative splicing to chromatin. The structure of the apo-MRG15 MRG domain implicated in interactions with diverse proteins has been described, but not in complex with any of its targets. Here, we structurally and functionally characterize the interaction between MRG15 and Pf1, two constitutively associated subunits of the histone deacetylase-associated Rpd3S/Sin3S corepressor complex. The MRG domain adopts a structure reminiscent of the apo state, whereas the Pf1 MRG-binding domain engages two discrete hydrophobic surfaces on the MRG domain via a bipartite motif comprising an α-helix and a segment in an extended conformation, both of which are critical for high-affinity interactions. Multiple MRG15 interactors share an FxLP motif in the extended segment, but equivalent sequence/helical motifs are not readily evident, implying potential diversity in MRG-recognition mechanisms.Display Omitted► A nanomolar affinity complex formed by the seemingly promiscuous MRG15 MRG domain ► A predominantly hydrophobic interaction involving two discrete MRG surfaces ► An FxLP motif shared with multiple MRG-interactors engages one of the surfaces ► A helical motif absent in other interactors engages the MRG-dimerization interface
Modular Evolution and the Origins of Symmetry: Reconstruction of a Three-Fold Symmetric Globular Protein by Aron Broom; Andrew C. Doxey; Yuri D. Lobsanov; Lisa G. Berthin; David R. Rose; P. Lynne Howell; Brendan J. McConkey; Elizabeth M. Meiering (161-171).
The high frequency of internal structural symmetry in common protein folds is presumed to reflect their evolutionary origins from the repetition and fusion of ancient peptide modules, but little is known about the primary sequence and physical determinants of this process. Unexpectedly, a sequence and structural analysis of symmetric subdomain modules within an abundant and ancient globular fold, the β-trefoil, reveals that modular evolution is not simply a relic of the ancient past, but is an ongoing and recurring mechanism for regenerating symmetry, having occurred independently in numerous existing β-trefoil proteins. We performed a computational reconstruction of a β-trefoil subdomain module and repeated it to form a newly three-fold symmetric globular protein, ThreeFoil. In addition to its near perfect structural identity between symmetric modules, ThreeFoil is highly soluble, performs multivalent carbohydrate binding, and has remarkably high thermal stability. These findings have far-reaching implications for understanding the evolution and design of proteins via subdomain modules.Display Omitted► Recurring evolution of symmetric beta-trefoils via subdomain module repetition ► Consensus and computational reconstruction of threefold symmetric globular protein ► Reconstructed symmetric beta-trefoil is well-folded, functional, and highly stable ► Numerous subdomain repetition events in proteins with multivalent binding functions
Assembly of the Eukaryotic PLP-Synthase Complex from Plasmodium and Activation of the Pdx1 Enzyme by Gabriela Guédez; Katharina Hipp; Volker Windeisen; Bianca Derrer; Martin Gengenbacher; Bettina Böttcher; Irmgard Sinning; Barbara Kappes; Ivo Tews (172-184).
Biosynthesis of vitamins is fundamental to malaria parasites. Plasmodia synthesize the active form of vitamin B6 (pyridoxal 5′-phosphate, PLP) using a PLP synthase complex. The EM analysis shown here reveals a random association pattern of up to 12 Pdx2 glutaminase subunits to the dodecameric Pdx1 core complex. Interestingly, Plasmodium falciparum PLP synthase organizes in fibers. The crystal structure shows differences in complex formation to bacterial orthologs as interface variations. Alternative positioning of an α helix distinguishes an open conformation from a closed state when the enzyme binds substrate. The pentose substrate is covalently attached through its C1 and forms a Schiff base with Lys84. Ammonia transfer between Pdx2 glutaminase and Pdx1 active sites is regulated by a transient tunnel. The mutagenesis analysis allows defining the requirement for conservation of critical methionines, whereas there is also plasticity in ammonia tunnel construction as seen from comparison across different species.Display Omitted► Plasmodium falciparum PLP synthase varies in composition and can form fibers ► Optimizing challenging 3D crystals using cross-species chimeric complexes ► A helical switch activation in the covalent Pdx1-ribose substrate complex ► Functional analysis of methionines critical in ammonia transfer
Structural Basis for Recognition of H3T3ph and Smac/DIABLO N-terminal Peptides by Human Survivin by Jiamu Du; Alexander E. Kelly; Hironori Funabiki; Dinshaw J. Patel (185-195).
Survivin is an inhibitor of apoptosis family protein implicated in apoptosis and mitosis. In apoptosis, it has been shown to recognize the Smac/DIABLO protein. It is also a component of the chromosomal passenger complex, a key player during mitosis. Recently, Survivin was identified in vitro and in vivo as the direct binding partner for phosphorylated Thr3 on histone H3 (H3T3ph). We have undertaken structural and binding studies to investigate the molecular basis underlying recognition of H3T3ph and Smac/DIABLO N-terminal peptides by Survivin. Our crystallographic studies establish recognition of N-terminal Ala in both complexes and identify intermolecular hydrogen-bonding interactions in the Survivin phosphate-binding pocket that contribute to H3T3ph mark recognition. In addition, our calorimetric data establish that Survivin binds tighter to the H3T3ph-containing peptide relative to the N-terminal Smac/DIABLO peptide, and this preference can be reversed through structure-guided mutations that increase the hydrophobicity of the phosphate-binding pocket.► Survivin primarily recognizes N-terminal Ala1 of H3, H3T3ph, and SmacN peptides ► The binding preference is determined by Lys62 and His80 side chains of Survivin ► Survivin exhibits a 25-fold preference for H3T3ph over SamcN peptide ► K62Y/H80W Survivin mutant reverses specificity in favor of SmacN peptide