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The secondary Na+/citrate symporter CitS of Klebsiella pneumoniae is the best-characterized member of the 2-hydroxycarboxylate transporter family. The recent projection structure gave insight into its overall structural organization. Here, we present the three-dimensional map of dimeric CitS obtained with electron crystallography. Each monomer has 13 a-helical transmembrane segments; six are organized in a distal helix cluster and seven in the central dimer interface domain. Based on structural analyses and comparison to VcINDY, we propose a molecular model for CitS, assign the helices, and demonstrate the internal structural symmetry. We also present projections of CitS in several conformational states induced by the presence and absence of sodium and citrate as substrates. Citrate binding induces a defined movement of a helices within the distal helical cluster. Based on this, we propose a substrate translocation site and conformational changes that are in agreement with the transport model of ‘‘alternating access’’.

Dysregulation of apoptosis is associated with several human diseases. The main apoptotic mediators are caspases, which propagate death signals to downstream targets. Executioner caspase-3 is responsible for the majority of cleavage events and its therapeutic potential is of high interest with to date several available active site peptide inhibitors. These molecules inhibit caspase-3, but also homologous caspases. Here, we describe caspase-3 specific inhibitors D3.4 and D3.8, which have been selected from a library of designed ankyrin repeat proteins (DARPins). The crystal structures of D3.4 and mutants thereof show how high specificity and inhibition is achieved. They also show similarities in the binding mode with that of the natural caspase inhibitor XIAP (X-linked inhibitor of apoptosis). The kinetic data reveal a competitive inhibition mechanism. D3.4 is specific for caspase-3 and does not bind the highly homologous caspase-7. D3.4 therefore is an excellent tool to define the precise role of caspase-3 in the various apoptotic pathways., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The β-aminopeptidase BapA from Sphingosinicella xenopeptidilytica belongs to the N-terminal nucleophile (Ntn) hydrolases of the DmpA-like family and has the unprecedented property of cleaving N-terminal β-amino acid residues from peptides. We determined the crystal structures of the native (αβ)₄ heterooctamer and of the 153 kDa precursor homotetramer at a resolution of 1.45 and 1.8 Å, respectively. These structures together with mutational analyses strongly support mechanisms for autoproteolysis and catalysis that involve residues Ser250, Ser288, and Glu290. The autoproteolytic mechanism is different from the one so far described for Ntn hydrolases. The structures together with functional data also provide insight into the discriminating features of the active site cleft that determine substrate specificity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Sphingosine-1-phosphate lyase (SPL), a key enzyme of sphingolipid metabolism, catalyzes the irreversible degradation of sphingoid base phosphates. Its main substrate sphingosine-1-phosphate (S1P) acts both extracellularly, by binding G protein-coupled receptors of the lysophospholipid receptor family, and inside the cell, as a second messenger. There, S1P takes part in regulating various cellular processes and its levels are tightly regulated. SPL is a pivotal enzyme regulating S1P intracellular concentrations and a promising drug target for the design of immunosuppressants. We structurally and functionally characterized yeast SPL (Dpl1p) and its first prokaryotic homolog, from Symbiobacterium thermophilum. The Dpl1p structure served as a basis for a very reliable model of Homo sapiens SPL. The above results, together with in vitro and in vivo studies of SPL mutants, reveal which residues are involved in activity and substrate binding and pave the way to studies aimed at controlling the activity of this pivotal enzyme., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Caspases are proteases with an active-site cysteine and aspartate specificity in their substrates. They are involved in apoptotic cell death and inflammation, and dysfunction of these enzymes is directly linked to a variety of diseases. Caspase-8 initiates an apoptotic pathway triggered by external stimuli. It was previously characterized in its active inhibitor bound state by crystallography. Here we present the solution structure of the monomeric unprocessed catalytic domain of the caspase-8 zymogen, procaspase-8, showing for the first time the position of the linker and flexibility of the active site forming loops. Biophysical studies of carefully designed mutants allowed disentangling dimerization and processing, and we could demonstrate lack of activity of monomeric uncleaved procaspase-8 and of a processed but dimerization-incompetent mutant. The data provide experimental support in so-far unprecedented detail, and reveal why caspase-8 (and most likely other initiator caspases) needs the dimerization platform during activation.

The structure of proteins that are difficult to crystallize can often be solved by forming a noncovalent complex with a helper protein--a crystallization "chaperone." Although several such applications have been described to date, their handling usually is still very laborious. A valuable addition to the present repertoire of binding proteins is the recently developed designed ankyrin repeat protein (DARPin) technology. DARPins are built based on the natural ankyrin repeat protein fold with randomized surface residue positions allowing specific binding to virtually any target protein. The broad potential of these binding proteins for X-ray crystallography is illustrated by five cocrystal structures that have been determined recently comprising target proteins from distinct families, namely a sugar binding protein, two kinases, a caspase, and a membrane protein. This article reviews the opportunities of this technology for structural biology and the structural aspects of the DARPin-protein complexes.

Adhesive type 1 pili from uropathogenic Escherichia coli strains are heat and denaturant resistant, filamentous protein complexes. Individual pilus subunits associate through "donor strand complementation," whereby the incomplete immunoglobulin-like fold of each subunit is completed by the N-terminal extension of a neighboring subunit. We show that antiparallel donor strand insertion generally causes nonequilibrium behavior in protein folding and extreme activation energy barriers for dissociation of subunit-subunit complexes. We identify the most kinetically stable, noncovalent protein complex known to date. The complex between the pilus subunit FimG and the donor strand peptide of the subunit FimF shows an extrapolated dissociation half-life of 3 x 10(9) years. The 15 residue peptide forms ideal intermolecular beta sheet H-bonds with FimG over 10 residues, and its hydrophobic side chains strongly interact with the hydrophobic core of FimG. The results show that kinetic stability and nonequilibrium behavior in protein folding confers infinite stability against dissociation in extracellular protein complexes.

Specific and potent caspase inhibitors are indispensable for the dissection of the intricate pathways leading to apoptosis. We selected a designed ankyrin repeat protein (DARPin) from a combinatorial library that inhibits caspase-2 in vitro with a subnanomolar inhibition constant and, in contrast to the peptidic caspase inhibitors, with very high specificity for this particular caspase. The crystal structure of this inhibitor (AR_F8) in complex with caspase-2 reveals the molecular basis for the specificity and, together with kinetic analyses, the allosteric mechanism of inhibition. The structure also shows a conformation of the active site that can be exploited for the design of inhibitory compounds. AR_F8 is a specific inhibitor of an initiator caspase and has the potential to help identify the function of caspase-2 in the complex biological apoptotic signaling network.

Dimethylarginine dimethylaminohydrolase (DDAH) is involved in the regulation of nitric oxide synthase (NOS) by metabolizing the free endogenous arginine derivatives N(omega)-methyl-L-arginine (MMA) and N(omega),N(omega)-dimethyl-L-arginine (ADMA), which are competitive inhibitors of NOS. Here, we present high-resolution crystal structures of DDAH isoform 1 (DDAH-1) isolated from bovine brain in complex with different inhibitors, including S-nitroso-L-homocysteine and Zn2+, a regulator of this mammalian enzyme. The structure of DDAH-1 consists of a propeller-like fold similar to other arginine-modifying enzymes and a flexible loop, which adopts different conformations and acts as a lid at the entrance of the active site. The orientation and interaction mode of inhibitors in the active site give insight into the regulation and the molecular mechanism of the enzyme. The presented structures provide a basis for the structure-based development of specific DDAH-1 inhibitors that might be useful in the therapeutic treatment of NOS dysfunction-related diseases.

Aminoglycoside phosphotransferase (3')-IIIa (APH) is a bacterial kinase that confers antibiotic resistance to many pathogenic bacteria and shares structural homology with eukaryotic protein kinases. We report here the crystal structure of APH, trapped in an inactive conformation by a tailor-made inhibitory ankyrin repeat (AR) protein, at 2.15 A resolution. The inhibitor was selected from a combinatorial library of designed AR proteins. The AR protein binds the C-terminal lobe of APH and thereby stabilizes three alpha helices, which are necessary for substrate binding, in a significantly displaced conformation. BIAcore analysis and kinetic enzyme inhibition experiments are consistent with the proposed allosteric inhibition mechanism. In contrast to most small-molecule kinase inhibitors, the AR proteins are not restricted to active site binding, allowing for higher specificity. Inactive conformations of pharmaceutically relevant enzymes, as can be elucidated with the approach presented here, represent powerful starting points for rational drug design.

DsbD from Escherichia coli transports two electrons from cytoplasmic thioredoxin to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of an N-terminal periplasmic domain (nDsbD), a C-terminal periplasmic domain, and a central transmembrane domain. Each domain possesses two cysteines required for electron transport. Herein, we demonstrate fast (3.9 x 10(5) M(-1)s(-1)) and direct disulfide exchange between nDsbD and CcmG, a highly specific disulfide reductase essential for cytochrome c maturation. We determined the crystal structure of the disulfide-linked complex between nDsbD and the soluble part of CcmG at 1.94 A resolution. In contrast to the other two known complexes of nDsbD with target proteins, the N-terminal segment of nDsbD contributes to specific recognition of CcmG. This and other features, like the possibility of using an additional interaction surface, constitute the structural basis for the adaptability of nDsbD to different protein substrates.

Signaling in apoptosis and inflammation is often mediated by proteins of the death domain superfamily in the Fas/FADD/Caspase-8 or the Apaf-1/Caspase-9 pathways. This superfamily currently comprises the death domain (DD), death effector domain (DED), caspase recruitment domain (CARD), and pyrin domain (PYD) subfamilies. The PYD subfamily is most abundant, but three-dimensional structures are only available for the subfamilies DD, DED, and CARD, which have an antiparallel arrangement of six alpha helices as common fold. This paper presents the NMR structure of PYD of NALP1, a protein that is involved in the innate immune response and is a component of the inflammasome. The structure of NALP1 PYD differs from all other known death domain superfamily structures in that the third alpha helix is replaced by a flexibly disordered loop. This unique feature appears to relate to the molecular basis of familial Mediterranean fever (FMF), a genetic disease caused by single-point mutations.