Design of High Affinity Binders to Convex Protein Target Sites.

While there has been progress in the de novo design of small globular miniproteins (50-65 residues) to bind to primarily concave regions of a target protein surface, computational design of minibinders to convex binding sites remains an outstanding challenge due to low level of overall shape complementarity. Here, we describe a general approach to generate computationally designed proteins which bind to convex target sites that employ geometrically matching concave scaffolds. We used this approach to design proteins binding to TGFßRII, CTLA-4 and PD-L1 which following experimental optimization have low nanomolar to picomolar affinities and potent biological activity. Co-crystal structures of the TGFßRII and CTLA-4 binders in complex with the receptors are in close agreement with the design models. Our approach provides a general route to generating very high affinity binders to convex protein target sites.

A hemoprotein with a zinc-mirror heme site ties heme availability to carbon metabolism in cyanobacteria.

Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site. Together with structure-guided in vitro and in vivo experiments, we further demonstrate the existence of a functional link between heme binding by Dri1 (Domain related to iron 1, formerly ssr1698) and post-translational regulation of succinate dehydrogenase in the cyanobacterium Synechocystis, suggesting an iron-dependent regulatory link between photosynthesis and respiration. Given the ubiquity of proteins containing homologous domains and connections to heme metabolism across eukaryotes and prokaryotes, we propose that DRI (Domain Related to Iron; formerly DUF2470) functions at the molecular level as a heme-dependent regulatory domain.

Phosphate Binding in PNP Alters Transition-State Analogue Affinity and Subunit Cooperativity.

Purine nucleoside phosphorylases (PNPs) catalyze the phosphorolysis of 6-oxypurine nucleosides with an HPO42- dianion nucleophile. Nucleosides and phosphate occupy distinct pockets in the PNP active site. Evaluation of the HPO42- site by mutagenesis, cooperative binding studies, and thermodynamic and structural analysis demonstrate that alterations in the HPO42- binding site can render PNP inactive and significantly impact subunit cooperativity and binding to transition-state analogue inhibitors. Cooperative interactions between the cationic transition-state analogue and the anionic HPO42- nucleophile demonstrate the importance of reforming the transition-state ensemble for optimal inhibition with transition-state analogues. Altered phosphate binding in the catalytic site mutants helps to explain one of the known lethal PNP deficiency syndromes in humans.

Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection.

Andes virus (ANDV) and Sin Nombre virus (SNV) are the etiologic agents of severe hantavirus cardiopulmonary syndrome (HCPS) in the Americas for which no FDA-approved countermeasures are available. Protocadherin-1 (PCDH1), a cadherin-superfamily protein recently identified as a critical host factor for ANDV and SNV, represents a new antiviral target; however, its precise role remains to be elucidated. Here, we use computational and experimental approaches to delineate the binding surface of the hantavirus glycoprotein complex on PCDH1's first extracellular cadherin repeat domain. Strikingly, a single amino acid residue in this PCDH1 surface influences the host species-specificity of SNV glycoprotein-PCDH1 interaction and cell entry. Mutation of this and a neighboring residue substantially protects Syrian hamsters from pulmonary disease and death caused by ANDV. We conclude that PCDH1 is a bona fide entry receptor for ANDV and SNV whose direct interaction with hantavirus glycoproteins could be targeted to develop new interventions against HCPS.

Inhibition and Mechanism of Plasmodium falciparum Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase.

Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT) is essential for purine salvage of hypoxanthine into parasite purine nucleotides. Transition state analogue inhibitors of PfHGXPRT are characterized by kinetic analysis, thermodynamic parameters, and X-ray crystal structures. Compound 1, 9-deazaguanine linked to an acyclic ribocation phosphonate mimic, shows a kinetic Ki of 0.5 nM. Isothermal titration calorimetry (ITC) experiments of 1 binding to PfHGXPRT reveal enthalpically driven binding with negative cooperativity for the binding of two inhibitor molecules in the tetrameric enzyme. Crystal structures of 1 bound to PfHGXPRT define the hydrogen bond and ionic contacts to complement binding thermodynamics. Dynamics of ribosyl transfer from 5-phospho-α-d-ribosyl 1-pyrophosphate (PRPP) to hypoxanthine were examined by 18O isotope exchange at the bridging phosphoryl oxygen of PRPP pyrophosphate. Rotational constraints or short transition state lifetimes prevent torsional rotation and positional isotope exchange of bridging to nonbridging oxygen in the α-pyrophosphoryl group. Thermodynamic analysis of the transition state analogue and magnesium pyrophosphate binding reveal random and cooperative binding to PfHGXPRT, unlike the obligatory ordered reaction kinetics reported earlier for substrate kinetics.

Structural and Functional Characterization of a Biliverdin-Binding Near-Infrared Fluorescent Protein From the Serpin Superfamily.

Biliverdin-binding serpins (BBSs) are proteins that are responsible for coloration in amphibians and fluoresce in the near-infrared (NIR) spectral region. Here we produced the first functional recombinant BBS of the polka-dot treefrog Boana punctata (BpBBS), assembled with its biliverdin (BV) chromophore, and report its biochemical and photochemical characterization. We determined the crystal structure of BpBBS at 2.05 Å resolution, which demonstrated its structural homology to the mammalian protease inhibitor alpha-1-antitrypsin. BV interaction with BpBBS was studied and it was found that the N-terminal polypeptide (residues 19-50) plays a critical role in the BV binding. By comparing BpBBS with the available NIR fluorescent proteins and expressing it in mammalian cells, we demonstrated its potential as a NIR imaging probe. These results provide insight into the non-inhibitory function of serpins, provide a basis for improving their performance in mammalian cells, and suggest possible paths for the development of BBS-based fluorescent probes.

Mechanism of Triphosphate Hydrolysis by Human MAT2A at 1.07 Å Resolution.

Human methionine adenosyltransferase MAT2A provides S-adenosyl-l-methionine (AdoMet) for methyl-transfer reactions. Epigenetic methylations influence expression patterns in development and in cancer. Transition-state analysis and kinetic studies have described the mechanism of AdoMet and triphosphate formation at the catalytic site. Hydrolysis of triphosphate to pyrophosphate and phosphate by MAT2A is required for product release and proceeds through a second chemical transition state. Crystal structures of MAT2A with analogues of AdoMet and pyrophosphate were obtained in the presence of Mg2+, Al3+, and F-. MgF3- is trapped as a PO3- mimic in a structure with malonate filling the pyrophosphate site. NMR demonstrates that MgF3- and AlF30 are bound by MAT2A as mimics of the departing phosphoryl group. Crystallographic analysis reveals a planar MgF3- acting to mimic a phosphoryl (PO3-) leaving group. The modeled transition state with PO3- has the phosphorus atom sandwiched symmetrically and equidistant (approximately 2 Å) between a pyrophosphate oxygen and the water nucleophile. A catalytic site arginine directs the nucleophilic water to the phosphoryl leaving group. The catalytic geometry of the transition-state reconstruction predicts a loose transition state with characteristics of symmetric nucleophilic displacement.

Mechanism and Inhibition of Human Methionine Adenosyltransferase 2A.

S-Adenosyl-l-methionine (AdoMet) is synthesized by the MAT2A isozyme of methionine adenosyltransferase in most human tissues and in cancers. Its contribution to epigenetic control has made it a target for anticancer intervention. A recent kinetic isotope effect analysis of MAT2A demonstrated a loose nucleophilic transition state. Here we show that MAT2A has a sequential mechanism with a rate-limiting step of formation of AdoMet, followed by rapid hydrolysis of the ß-γ bond of triphosphate, and rapid release of phosphate and pyrophosphate. MAT2A catalyzes the slow hydrolysis of both ATP and triphosphate in the absence of other reactants. Positional isotope exchange occurs with 18O as the 5'-oxygen of ATP. Loss of the triphosphate is sufficiently reversible to permit rotation and recombination of the α-phosphoryl group of ATP. Adenosine (α-ß or ß-γ)-imido triphosphates are slow substrates, and the respective imido triphosphates are inhibitors. The hydrolytically stable (α-ß, ß-γ)-diimido triphosphate (PNPNP) is a nanomolar inhibitor. The MAT2A protein structure is highly stabilized against denaturation by binding of PNPNP. A crystal structure of MAT2A with 5'-methylthioadenosine and PNPNP shows the ligands arranged appropriately in the ATP binding site. Two magnesium ions chelate the α- and γ-phosphoryl groups of PNPNP. The ß-phosphoryl oxygen is in contact with an essential potassium ion. Imidophosphate derivatives provide contact models for the design of catalytic site ligands for MAT2A.

Functional assignment of multiple catabolic pathways for D-apiose.

Colocation of the genes encoding ABC, TRAP, and TCT transport systems and catabolic pathways for the transported ligand provides a strategy for discovering novel microbial enzymes and pathways. We screened solute-binding proteins (SBPs) for ABC transport systems and identified three that bind D-apiose, a branched pentose in the cell walls of higher plants. Guided by sequence similarity networks (SSNs) and genome neighborhood networks (GNNs), the identities of the SBPs enabled the discovery of four catabolic pathways for D-apiose with eleven previously unknown reactions. The new enzymes include D-apionate oxidoisomerase, which catalyzes hydroxymethyl group migration, as well as 3-oxo-isoapionate-4-phosphate decarboxylase and 3-oxo-isoapionate-4-phosphate transcarboxylase/hydrolase, which are RuBisCO-like proteins (RLPs). The web tools for generating SSNs and GNNs are publicly accessible ( http://efi.igb.illinois.edu/efi-est/ ), so similar 'genomic enzymology' strategies for discovering novel pathways can be used by the community.

Structures of the L27 Domain of Disc Large Homologue 1 Protein Illustrate a Self-Assembly Module.

Disc large 1 (Dlg1) proteins, members of the MAGUK protein family, are linked to cell polarity via their participation in multiprotein assemblies. At their N-termini, Dlg1 proteins contain a L27 domain. Typically, the L27 domains participate in the formation of obligate hetero-oligomers with the L27 domains from their cognate partners. Among the MAGUKs, Dlg1 proteins exist as homo-oligomers, and the oligomerization is solely dependent on the L27 domain. Here we provide biochemical and structural evidence of homodimerization via the L27 domain of Dlg1 from Drosophila melanogaster. The structure reveals that the core of the dimer is formed by a distinctive six-helix assembly, involving all three conserved helices from each subunit (monomer). The homodimer interface is extended by the C-terminal tail of the L27 domain of Dlg1, which forms a two-stranded antiparallel ß-sheet. The structure reconciles and provides a structural context for a large body of available mutational data. From our analyses, we conclude that the observed L27 homodimerization is most likely a feature unique to the Dlg1 orthologs within the MAGUK family.

Genetic and structural analysis of the essential fission yeast RNA polymerase II CTD phosphatase Fcp1.

Protein phosphatases regulate mRNA synthesis and processing by remodeling the carboxy-terminal domain (CTD) of RNA polymerase II (Pol2) to dynamically inscribe a Pol2 CTD code. Fission yeast Fcp1 (SpFcp1) is an essential 723-amino acid CTD phosphatase that preferentially hydrolyzes Ser2-PO4 of the YS(2)PTSPS repeat. The SpFcp1 catalytic domain (aa 140-580) is composed of a DxDxT acyl-phosphatase module (FCPH) and a BRCT module. Here we conducted a genetic analysis of SpFcp1, which shows that (i) phosphatase catalytic activity is required for vegetative growth of fission yeast; (ii) the flanking amino-terminal domain (aa 1-139) and its putative metal-binding motif C(99)H(101)Cys(109)C(112) are essential; (iii) the carboxy-terminal domain (aa 581-723) is dispensable; (iv) a structurally disordered internal segment of the FCPH domain (aa 330-393) is dispensable; (v) lethal SpFcp1 mutations R271A and R299A are rescued by shortening the Pol2 CTD repeat array; and (vi) CTD Ser2-PO4 is not the only essential target of SpFcp1 in vivo. Recent studies highlight a second CTD code involving threonine phosphorylation of a repeat motif in transcription elongation factor Spt5. We find that Fcp1 can dephosphorylate Thr1-PO4 of the fission yeast Spt5 CTD nonamer repeat T(1)PAWNSGSK. We identify Arg271 as a governor of Pol2 versus Spt5 CTD substrate preference. Our findings implicate Fcp1 as a versatile sculptor of both the Pol2 and Spt5 CTD codes. Finally, we report a new 1.45 Å crystal structure of SpFcp1 with Mg(2+) and AlF3 that mimics an associative phosphorane transition state of the enzyme-aspartyl-phosphate hydrolysis reaction.

Danio rerio αE-catenin is a monomeric F-actin binding protein with distinct properties from Mus musculus αE-catenin.

It is unknown whether homologs of the cadherin·catenin complex have conserved structures and functions across the Metazoa. Mammalian αE-catenin is an allosterically regulated actin-binding protein that binds the cadherin·ß-catenin complex as a monomer and whose dimerization potentiates F-actin association. We tested whether these functional properties are conserved in another vertebrate, the zebrafish Danio rerio. Here we show, despite 90% sequence identity, that Danio rerio and Mus musculus αE-catenin have striking functional differences. We demonstrate that D. rerio αE-catenin is monomeric by size exclusion chromatography, native PAGE, and small angle x-ray scattering. D. rerio αE-catenin binds F-actin in cosedimentation assays as a monomer and as an α/ß-catenin heterodimer complex. D. rerio αE-catenin also bundles F-actin, as shown by negative stained transmission electron microscopy, and does not inhibit Arp2/3 complex-mediated actin nucleation in bulk polymerization assays. Thus, core properties of α-catenin function, F-actin and ß-catenin binding, are conserved between mouse and zebrafish. We speculate that unique regulatory properties have evolved to match specific developmental requirements.

Structural insights to how mammalian capping enzyme reads the CTD code.

Physical interaction between the phosphorylated RNA polymerase II carboxyl-terminal domain (CTD) and cellular capping enzymes is required for efficient formation of the 5' mRNA cap, the first modification of nascent mRNA. Here, we report the crystal structure of the RNA guanylyltransferase component of mammalian capping enzyme (Mce) bound to a CTD phosphopeptide. The CTD adopts an extended ß-like conformation that docks Tyr1 and Ser5-PO(4) onto the Mce nucleotidyltransferase domain. Structure-guided mutational analysis verified that the Mce-CTD interface is a tunable determinant of CTD binding and stimulation of guanylyltransferase activity, and of Mce function in vivo. The location and composition of the CTD binding site on mammalian capping enzyme is distinct from that of a yeast capping enzyme that recognizes the same CTD primary structure. Thus, capping enzymes from different taxa have evolved different strategies to read the CTD code.

Enzymology of RNA cap synthesis.

The 5' guanine-N7 methyl cap is unique to cellular and viral messenger RNA (mRNA) and is the first co-transcriptional modification of mRNA. The mRNA cap plays a pivotal role in mRNA biogenesis and stability, and is essential for efficient splicing, mRNA export, and translation. Capping occurs by a series of three enzymatic reactions that results in formation of N7-methyl guanosine linked through a 5'-5' inverted triphosphate bridge to the first nucleotide of a nascent transcript. Capping of cellular mRNA occurs co-transcriptionally and in vivo requires that the capping apparatus be physically associated with the RNA polymerase II elongation complex. Certain capped mRNAs undergo further methylation to generate distinct cap structures. Although mRNA capping is conserved among viruses and eukaryotes, some viruses have adopted strategies for capping mRNA that are distinct from the cellular mRNA capping pathway.

The structure of Fcp1, an essential RNA polymerase II CTD phosphatase.

Kinases and phosphatases regulate mRNA synthesis and processing by phosphorylating and dephosphorylating the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. Fcp1 is an essential CTD phosphatase that preferentially hydrolyzes Ser2-PO(4) of the tandem YSPTSPS CTD heptad array. Fcp1 crystal structures were captured at two stages of the reaction pathway: a Mg-BeF(3) complex that mimics the aspartylphosphate intermediate and a Mg-AlF(4)(-) complex that mimics the transition state of the hydrolysis step. Fcp1 is a Y-shaped protein composed of an acylphosphatase domain located at the base of a deep canyon formed by flanking modules that are missing from the small CTD phosphatase (SCP) clade: an Fcp1-specific helical domain and a C-terminal BRCA1 C-terminal (BRCT) domain. The structure and mutational analysis reveals that Fcp1 and Scp1 (a Ser5-selective phosphatase) adopt different CTD-binding modes; we surmise the CTD threads through the Fcp1 canyon to access the active site.

Mutational analysis of Encephalitozoon cuniculi mRNA cap (guanine-N7) methyltransferase, structure of the enzyme bound to sinefungin, and evidence that cap methyltransferase is the target of sinefungin's antifungal activity.

Cap (guanine-N7) methylation is an essential step in eukaryal mRNA synthesis and a potential target for antiviral, antifungal, and antiprotozoal drug discovery. Previous mutational and structural analyses of Encephalitozoon cuniculi Ecm1, a prototypal cellular cap methyltransferase, identified amino acids required for cap methylation in vivo, but also underscored the nonessentiality of many side chains that contact the cap and AdoMet substrates. Here we tested new mutations in residues that comprise the guanine-binding pocket, alone and in combination. The outcomes indicate that the shape of the guanine binding pocket is more crucial than particular base edge interactions, and they highlight the contributions of the aliphatic carbons of Phe-141 and Tyr-145 that engage in multiple van der Waals contacts with guanosine and S-adenosylmethionine (AdoMet), respectively. We purified 45 Ecm1 mutant proteins and assayed them for methylation of GpppA in vitro. Of the 21 mutations that resulted in unconditional lethality in vivo,14 reduced activity in vitro to < or = 2% of the wild-type level and 5 reduced methyltransferase activity to between 4 and 9% of wild-type Ecm1. The natural product antibiotic sinefungin is an AdoMet analog that inhibits Ecm1 with modest potency. The crystal structure of an Ecm1-sinefungin binary complex reveals sinefungin-specific polar contacts with main-chain and side-chain atoms that can explain the 3-fold higher affinity of Ecm1 for sinefungin versus AdoMet or S-adenosylhomocysteine (AdoHcy). In contrast, sinefungin is an extremely potent inhibitor of the yeast cap methyltransferase Abd1, to which sinefungin binds 900-fold more avidly than AdoHcy or AdoMet. We find that the sensitivity of Saccharomyces cerevisiae to growth inhibition by sinefungin is diminished when Abd1 is overexpressed. These results highlight cap methylation as a principal target of the antifungal activity of sinefungin.

How guanylate-binding proteins achieve assembly-stimulated processive cleavage of GTP to GMP.

Interferons are immunomodulatory cytokines that mediate anti-pathogenic and anti-proliferative effects in cells. Interferon-gamma-inducible human guanylate binding protein 1 (hGBP1) belongs to the family of dynamin-related large GTP-binding proteins, which share biochemical properties not found in other families of GTP-binding proteins such as nucleotide-dependent oligomerization and fast cooperative GTPase activity. hGBP1 has an additional property by which it hydrolyses GTP to GMP in two consecutive cleavage reactions. Here we show that the isolated amino-terminal G domain of hGBP1 retains the main enzymatic properties of the full-length protein and can cleave GDP directly. Crystal structures of the N-terminal G domain trapped at successive steps along the reaction pathway and biochemical data reveal the molecular basis for nucleotide-dependent homodimerization and cleavage of GTP. Similar to effector binding in other GTP-binding proteins, homodimerization is regulated by structural changes in the switch regions. Homodimerization generates a conformation in which an arginine finger and a serine are oriented for efficient catalysis. Positioning of the substrate for the second hydrolysis step is achieved by a change in nucleotide conformation at the ribose that keeps the guanine base interactions intact and positions the beta-phosphates in the gamma-phosphate-binding site.

Crystal structure of IIGP1: a paradigm for interferon-inducible p47 resistance GTPases.

Interferon-inducible p47 GTPases are critical mediators of cell-autonomous resistance against several intracellular pathogens. Here we present the first crystal structure of a member of this novel GTPase family, IIGP1, in its nucleotide-free, GDP-, and GppNHp-bound form. The structure shows a Ras-like G domain between an N-terminal three-helix bundle and a complex system of C-terminal helices and loops. Sequence comparison and secondary structure prediction suggest the IIGP1 structure to be a valid model for the p47 GTPase family. The IIGP1 crystals contain a noncrystallographic dimer. We show that the dimer is required for cooperative GTP hydrolysis and GTP-dependent oligomerization of IIGP1. We also present the GDP- and GppNHp-bound monomeric structures of two dimer interface mutants. Our structures direct approaches to the analysis of the catalytic mechanism of IIGP1 and provide a coherent basis for structure-function studies aimed at elucidating the mechanistic basis of pathogen resistance caused by these enigmatic GTPases.