Novel variants provide differential stabilisation of human equilibrative nucleoside transporter 1 states.

Human equilibrative nucleoside transporters represent a major pharmaceutical target for cardiac, cancer and viral therapies. Understanding the molecular basis for transport is crucial for the development of improved therapeutics through structure-based drug design. ENTs have been proposed to utilise an alternating access mechanism of action, similar to that of the major facilitator superfamily. However, ENTs lack functionally-essential features of that superfamily, suggesting that they may use a different transport mechanism. Understanding the molecular basis of their transport requires insight into diverse conformational states. Differences between intermediate states may be discrete and mediated by subtle gating interactions, such as salt bridges. We identified four variants of human equilibrative nucleoside transporter isoform 1 (hENT1) at the large intracellular loop (ICL6) and transmembrane helix 7 (TM7) that stabilise the apo-state (∆T m 0.7-1.5°C). Furthermore, we showed that variants K263A (ICL6) and I282V (TM7) specifically stabilise the inhibitor-bound state of hENT1 (∆∆T m 5.0 ± 1.7°C and 3.0 ± 1.8°C), supporting the role of ICL6 in hENT1 gating. Finally, we showed that, in comparison with wild type, variant T336A is destabilised by nitrobenzylthioinosine (∆∆T m -4.7 ± 1.1°C) and binds it seven times worse. This residue may help determine inhibitor and substrate sensitivity. Residue K263 is not present in the solved structures, highlighting the need for further structural data that include the loop regions.

Unexpected structures formed by the kinase RET C634R mutant extracellular domain suggest potential oncogenic mechanisms in MEN2A.

The RET receptor tyrosine kinase plays a pivotal role in cell survival, proliferation, and differentiation, and its abnormal activation leads to cancers through receptor fusions or point mutations. Mutations that disrupt the disulfide network in the extracellular domain (ECD) of RET drive multiple endocrine neoplasia type 2A (MEN2A), a hereditary syndrome associated with the development of thyroid cancers. However, structural details of how specific mutations affect RET are unclear. Here, we present the first structural insights into the ECD of the RET(C634R) mutant, the most common mutation in MEN2A. Using electron microscopy, we demonstrate that the C634R mutation causes ligand-independent dimerization of the RET ECD, revealing an unusual tail-to-tail conformation that is distinct from the ligand-induced signaling dimer of WT RET. Additionally, we show that the RETC634R ECD dimer can form complexes with at least two of the canonical RET ligands and that these complexes form very different structures than WT RET ECD upon ligand binding. In conclusion, this structural analysis of cysteine-mutant RET ECD suggests a potential key mechanism of cancer induction in MEN2A, both in the absence and presence of its native ligands, and may offer new targets for therapeutic intervention.

Tying up the Loose Ends: A Mathematically Knotted Protein.

Knots have attracted scientists in mathematics, physics, biology, and engineering. Long flexible thin strings easily knot and tangle as experienced in our daily life. Similarly, long polymer chains inevitably tend to get trapped into knots. Little is known about their formation or function in proteins despite >1,000 knotted proteins identified in nature. However, these protein knots are not mathematical knots with their backbone polypeptide chains because of their open termini, and the presence of a "knot" depends on the algorithm used to create path closure. Furthermore, it is generally not possible to control the topology of the unfolded states of proteins, therefore making it challenging to characterize functional and physicochemical properties of knotting in any polymer. Covalently linking the amino and carboxyl termini of the deeply trefoil-knotted YibK from Pseudomonas aeruginosa allowed us to create the truly backbone knotted protein by enzymatic peptide ligation. Moreover, we produced and investigated backbone cyclized YibK without any knotted structure. Thus, we could directly probe the effect of the backbone knot and the decrease in conformational entropy on protein folding. The backbone cyclization did not perturb the native structure and its cofactor binding affinity, but it substantially increased the thermal stability and reduced the aggregation propensity. The enhanced stability of a backbone knotted YibK could be mainly originated from an increased ruggedness of its free energy landscape and the destabilization of the denatured state by backbone cyclization with little contribution from a knot structure. Despite the heterogeneity in the side-chain compositions, the chemically unfolded cyclized YibK exhibited several macroscopic physico-chemical attributes that agree with theoretical predictions derived from polymer physics.

Functional Characterization of the γ-Aminobutyric Acid Transporter from Mycobacterium smegmatis MC2 155 Reveals Sodium-Driven GABA Transport.

Characterizing the mycobacterial transporters involved in the uptake and/or catabolism of host-derived nutrients required by mycobacteria may identify novel drug targets against tuberculosis. Here, we identify and characterize a member of the amino acid-polyamine-organocation superfamily, a potential γ-aminobutyric acid (GABA) transport protein, GabP, from Mycobacterium smegmatis The protein was expressed to a level allowing its purification to homogeneity, and size exclusion chromatography coupled with multiangle laser light scattering (SEC-MALLS) analysis of the purified protein showed that it was dimeric. We showed that GabP transported γ-aminobutyric acid both in vitro and when overexpressed in E. coli Additionally, transport was greatly reduced in the presence of ß-alanine, suggesting it could be either a substrate or inhibitor of GabP. Using GabP reconstituted into proteoliposomes, we demonstrated that γ-aminobutyric acid uptake is driven by the sodium gradient and is stimulated by membrane potential. Molecular docking showed that γ-aminobutyric acid binds MsGabP, another Mycobacterium smegmatis putative GabP, and the Mycobacterium tuberculosis homologue in the same manner. This study represents the first expression, purification, and characterization of an active γ-aminobutyric acid transport protein from mycobacteria.IMPORTANCE The spread of multidrug-resistant tuberculosis increases its global health impact in humans. As there is transmission both to and from animals, the spread of the disease also increases its effects in a broad range of animal species. Identifying new mycobacterial transporters will enhance our understanding of mycobacterial physiology and, furthermore, provides new drug targets. Our target protein is the gene product of msmeg_6196, annotated as GABA permease, from Mycobacterium smegmatis strain MC2 155. Our current study demonstrates it is a sodium-dependent GABA transporter that may also transport ß-alanine. As GABA may well be an essential nutrient for mycobacterial metabolism inside the host, this could be an attractive target for the development of new drugs against tuberculosis.

Expression and purification of the extracellular domain of wild-type humanRET and the dimeric oncogenic mutant C634R.

The receptor tyrosine kinase RET is essential in a variety of cellular processes. RET gain-of-function is strongly associated with several cancers, notably multiple endocrine neoplasia type 2A (MEN 2A), while RET loss-of-function causes Hirschsprung's disease and Parkinson's disease. To investigate the activation mechanism of RET as well as to enable drug development, over-expressed recombinant protein is needed for in vitro functional and structural studies. By comparing insect and mammalian cells expression of the RET extracellular domain (RETECD), we showed that the expression yields of RETECD using both systems were comparable, but mammalian cells produced monomeric functional RETECD, whereas RETECD expressed in insect cells was non-functional and multimeric. This was most likely due to incorrect disulfide formation. By fusing an Fc tag to the C-terminus of RETECD, we were able to produce, in HEK293T cells, dimeric oncogenic RETECD (C634R) for the first time. The protein remained dimeric even after cleavage of the tag via the cysteine disulfide, as in full-length RET in the context of MEN 2A and related pathologies. Our work thus provides valuable tools for functional and structural studies of the RET signaling system and its oncogenic activation mechanisms.

Defining Dynamics of Membrane-Bound Pyrophosphatases by Experimental and Computational Single-Molecule FRET.

Membrane-bound pyrophosphatases couple the hydrolysis of inorganic pyrophosphate to the pumping of ions (sodium or protons) across a membrane in order to generate an electrochemical gradient. This class of membrane protein is widely conserved across plants, fungi, archaea, and bacteria, but absent in multicellular animals, making them a viable target for drug design against protozoan parasites such as Plasmodium falciparum. An excellent understanding of many of the catalytic states throughout the enzymatic cycle has already been afforded by crystallography. However, the dynamics and kinetics of the catalytic cycle between these static snapshots remain to be elucidated. Here, we employ single-molecule Förster resonance energy transfer (FRET) measurements to determine the dynamic range and frequency of conformations available to the enzyme in a lipid bilayer during the catalytic cycle. First, we explore issues related to the introduction of fluorescent dyes by cysteine mutagenesis; we discuss the importance of residue selection for dye attachment, and the balance between mutating areas of the protein that will provide useful dynamics while not altering highly conserved residues that could disrupt protein function. To complement and guide the experiments, we used all-atom molecular dynamics simulations and computational methods to estimate FRET efficiency distributions for dye pairs at different sites in different protein conformational states. We present preliminary single-molecule FRET data that points to insights about the binding modes of different membrane-bound pyrophosphatase substrates and inhibitors.

Binding of EphrinA5 to RET receptor tyrosine kinase: An in vitro study.

Eph/Ephrin signaling pathways are crucial in regulating a large variety of physiological processes during development, such as cell morphology, proliferation, migration and axonal guidance. EphrinA (efn-A) ligands, in particular, can be activated by EphA receptors at cell-cell interfaces and have been proposed to cause reverse signaling via RET receptor tyrosine kinase. Such association has been reported to mediate spinal motor axon navigation, but conservation of the interactive signaling pathway and the molecular mechanism of the interaction are unclear. Here, we found Danio rerio efn-A5b bound to Mus musculus EphA4 with high affinity, revealing structurally and functionally conserved EphA/efn-A signaling. Interestingly, we observed no interaction between efn-A5b and RET from zebrafish, unlike earlier cell-based assays. Their lack of association indicates how complex efn-A signaling is and suggests that there may be other molecules involved in efn-A5-induced RET signaling.

Zebrafish GDNF and its co-receptor GFRα1 activate the human RET receptor and promote the survival of dopaminergic neurons in vitro.

Glial cell line-derived neurotrophic factor (GDNF) is a ligand that activates, through co-receptor GDNF family receptor alpha-1 (GFRα1) and receptor tyrosine kinase "RET", several signaling pathways crucial in the development and sustainment of multiple neuronal populations. We decided to study whether non-mammalian orthologs of these three proteins have conserved their function: can they activate the human counterparts? Using the baculovirus expression system, we expressed and purified Danio rerio RET, and its binding partners GFRα1 and GDNF, and Drosophila melanogaster RET and two isoforms of co-receptor GDNF receptor-like. Our results report high-level insect cell expression of post-translationally modified and dimerized zebrafish RET and its binding partners. We also found that zebrafish GFRα1 and GDNF are comparably active as mammalian cell-produced ones. We also report the first measurements of the affinity of the complex to RET in solution: at least for zebrafish, the Kd for GFRα1-GDNF binding RET is 5.9 µM. Surprisingly, we also found that zebrafish GDNF as well as zebrafish GFRα1 robustly activated human RET signaling and promoted the survival of cultured mouse dopaminergic neurons with comparable efficiency to mammalian GDNF, unlike E. coli-produced human proteins. These results contradict previous studies suggesting that mammalian GFRα1 and GDNF cannot bind and activate non-mammalian RET and vice versa.

[Pyrophosphate in medicine]. / Pyrofosfaatti lääketieteessä.

In all organisms from bacteria to humans, specific hydrolases--pyrophosphatases--hydrolyse inorganic pyrophosphate to phosphate. Without this, DNA, RNA and protein synthesis stops. Pyrophosphatases are thus essential for all life. In humans, disorders in pyrophosphate metabolism cause chondrocalcinosis and hypophosphatasia. Currently, pyrophosphate analogues, e.g. alendronate, are in clinical use in osteoporosis and Paget's disease but also for e.g. complications of prostate cancer. In bacteria and protozoan parasites, membrane-bound pyrophosphatases (mPPases), which do not occur in humans, convert pyrophosphate to a proton or sodium gradient. mPPases, which are crucial for protozoan parasites, are thus promising drug targets e.g. for malaria and leishmaniasis.

Solution structure and biophysical characterization of the multifaceted signalling effector protein growth arrest specific-1.

BACKGROUND: The protein growth arrest specific-1 (GAS1) was discovered based on its ability to stop the cell cycle. During development it is involved in embryonic patterning, inhibits cell proliferation and mediates cell death, and has therefore been considered as a tumor suppressor. GAS1 is known to signal through two different cell membrane receptors: Rearranged during transformation (RET), and the sonic hedgehog receptor Patched-1. Sonic Hedgehog signalling is important in stem cell renewal and RET mediated signalling in neuronal survival. Disorders in both sonic hedgehog and RET signalling are connected to cancer progression. The neuroprotective effect of RET is controlled by glial cell-derived neurotrophic factor family ligands and glial cell-derived neurotrophic factor receptor alphas (GFRαs). Human Growth arrest specific-1 is a distant homolog of the GFRαs. RESULTS: We have produced and purified recombinant human GAS1 protein, and confirmed that GAS1 is a monomer in solution by static light scattering and small angle X-ray scattering analysis. The low resolution solution structure reveals that GAS1 is more elongated and flexible than the GFRαs, and the homology modelling of the individual domains show that they differ from GFRαs by lacking the amino acids for neurotrophic factor binding. In addition, GAS1 has an extended loop in the N-terminal domain that is conserved in vertebrates after the divergence of fishes and amphibians. CONCLUSIONS: We conclude that GAS1 most likely differs from GFRαs functionally, based on comparative structural analysis, while it is able to bind the extracellular part of RET in a neurotrophic factor independent manner, although with low affinity in solution. Our structural characterization indicates that GAS1 differs from GFRα's significantly also in its conformation, which probably reflects the functional differences between GAS1 and the GFRαs.

The structure of the NTPase that powers DNA packaging into Sulfolobus turreted icosahedral virus 2.

Biochemical reactions powered by ATP hydrolysis are fundamental for the movement of molecules and cellular structures. One such reaction is the encapsidation of the double-stranded DNA (dsDNA) genome of an icosahedrally symmetric virus into a preformed procapsid with the help of a genome-translocating NTPase. Such NTPases have been characterized in detail from both RNA and tailed DNA viruses. We present four crystal structures and the biochemical activity of a thermophilic NTPase, B204, from the nontailed, membrane-containing, hyperthermoacidophilic archaeal dsDNA virus Sulfolobus turreted icosahedral virus 2. These are the first structures of a genome-packaging NTPase from a nontailed, dsDNA virus with an archaeal host. The four structures highlight the catalytic cycle of B204, pinpointing the molecular movement between substrate-bound (open) and empty (closed) active sites. The protein is shown to bind both single-stranded and double-stranded nucleic acids and to have an optimum activity at 80°C and pH 4.5. The overall fold of B204 places it in the FtsK-HerA superfamily of P-loop ATPases, whose cellular and viral members have been suggested to share a DNA-translocating mechanism.

The structure and catalytic cycle of a sodium-pumping pyrophosphatase.

Membrane-integral pyrophosphatases (M-PPases) are crucial for the survival of plants, bacteria, and protozoan parasites. They couple pyrophosphate hydrolysis or synthesis to Na(+) or H(+) pumping. The 2.6-angstrom structure of Thermotoga maritima M-PPase in the resting state reveals a previously unknown solution for ion pumping. The hydrolytic center, 20 angstroms above the membrane, is coupled to the gate formed by the conserved Asp(243), Glu(246), and Lys(707) by an unusual "coupling funnel" of six α helices. Comparison with our 4.0-angstrom resolution structure of the product complex suggests that helix 12 slides down upon substrate binding to open the gate by a simple binding-change mechanism. Below the gate, four helices form the exit channel. Superimposing helices 3 to 6, 9 to 12, and 13 to 16 suggests that M-PPases arose through gene triplication.

Crystal structure and role of glycans and dimerization in folding of neuronal leucine-rich repeat protein AMIGO-1.

AMIGO-1 is the parent member of a novel family of three cell surface leucine-rich repeat (LRR) proteins. Its expression is induced by the binding of HMGB1 (high-mobility group box 1 protein) to RAGE (receptor for advanced glycation end products) on neurons. Binding of HMGB1 to RAGE is known to have a direct effect on cellular growth regulation and mobility, and AMIGO-1 directly supports growth of neuronal processes and fasciculation of neurites. In addition, the second member of the AMIGO-family, AMIGO-2, has been implicated in adhesion of tumor cells in adenocarcinoma and survival of neurons. We have determined the crystal structure of AMIGO-1 at 2.0 Å resolution, which reveals a typical cell surface LRR domain arrangement with N- and C-terminal capping domains with disulfide bridges, followed by a C2-type Ig domain. AMIGO-1 is a dimer, with the LRR regions forming the dimer interface, and sequence conservation analysis and static light-scattering measurements suggest that all three AMIGO family proteins form similar dimers. Based on the AMIGO-1 structure, we have also modeled AMIGO-2 and present small-angle X-ray scattering data on AMIGO-2 and AMIGO-3. Our mutagenesis studies show that AMIGO-1 dimerization is necessary for proper cell surface expression and thus probably for proper or stable folding in the endoplastic reticulum and for the function of the protein. Based on the data presented earlier, we also suggest that dimerization through the LRR-LRR interface is likely to be involved in cell-cell adhesion by AMIGO-1, while extensive glycosylation may have a role.

Structural determinants of growth factor binding and specificity by VEGF receptor 2.

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 22 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions.

Electrostatic interactions of Hsp-organizing protein tetratricopeptide domains with Hsp70 and Hsp90: computational analysis and protein engineering.

The Hsp-organizing protein (HOP) binds to the C termini of the chaperones Hsp70 and Hsp90, thus bringing them together so that substrate proteins can be passed from Hsp70 to Hsp90. Because Hsp90 is essential for the correct folding and maturation of many oncogenic proteins, it has become a significant target for anti-cancer drug design. HOP binds to Hsp70 and Hsp90 via two independent tetratricopeptide (TPR) domains, TPR1 and TPR2A, respectively. We have analyzed ligand binding using Poisson-Boltzmann continuum electrostatic calculations, free energy perturbation, molecular dynamics simulations, and site-directed mutagenesis to delineate the contribution of different interactions to the affinity and specificity of the TPR-peptide interactions. We found that continuum electrostatic calculations could be used to guide protein design by removing unfavorable interactions to increase binding affinity, with an 80-fold increase in affinity for TPR2A. Contributions at buried charged residues, however, were better predicted by free energy perturbation calculations. We suggest using a combination of the two approaches for increasing the accuracy of results, with free energy perturbation calculations used only at selected buried residues of the ligand binding pocket. Finally we present the crystal structure of TPR2A in complex with its non-cognate Hsp70 ligand, which provides insight on the origins of specificity in TPR domain-peptide recognition.

Comparison of GFL-GFRalpha complexes: further evidence relating GFL bend angle to RET signalling.

Glial cell line-derived neurotrophic factor (GDNF) activates the receptor tyrosine kinase RET by binding to the GDNF-family receptor alpha1 (GFRalpha1) and forming the GDNF(2)-GFRalpha1(2)-RET(2) heterohexamer complex. A previous crystal structure of the GDNF(2)-GFRalpha1(2) complex (PDB code 2v5e) suggested that differences in signalling in GDNF-family ligand (GFL) complexes might arise from differences in the bend angle between the two monomers in the GFL homodimer. Here, a 2.35 A resolution structure of the GDNF(2)-GFRalpha1(2) complex crystallized with new cell dimensions is reported. The structure was refined to a final R factor of 22.5% (R(free) = 28%). The structures of both biological tetrameric complexes in the asymmetric unit are very similar to 2v5e and different from the artemin-GFRalpha3 structure, even though there is a small change in the structure of the GDNF. By comparison of all known GDNF and artemin structures, it is concluded that GDNF is more bent and more flexible than artemin and that this may be related to RET signalling. Comparisons also suggest that the differences between artemin and GDNF arise from the increased curvature of the artemin ;fingers', which both increases the buried surface area in the monomer-monomer interface and changes the intermonomer bend angle. From sequence comparison, it is suggested that neuturin (the second GFL) adopts an artemin-like conformation, while persephin has a different conformation to the other three.

Recent inventions on receptor tyrosine kinase RET modulation.

Rearranged during transfection, RET, is a receptor tyrosine kinase expressed in neural crest derived cell lineages. RET is activated by dimerisation facilitated by its binding to the heterodimeric complex formed by Glial cell-derived neurotrophic factor (GDNF) -family ligand (GFL) and GNDF-family receptor (GFR). Both GDNFs and their co-receptors are a small protein family of four members. RET kinase mediated signaling can lead to survival, cell growth, differentiation, and migration. Pharmaceutically RET is of interest due to its involvement in several disease conditions. Oncogenic RET activation by mutations or rearragements predisposes to cancers like multiple endocrine neoplasia type 2 (A and B) and medullary thyroid carcinoma. Loss-of-function mutations in RET are a strong susceptibility factor for Hirschsprung disease, which is characterized by lack of ganglion cells in gastrointestinal tract. All the GFLs promote neuronal survival and GDNF is one of the most potent neurotrophic factors for dopaminergic neurons. Therefore, the neuroprotective capacity of RET activation to override the apoptotic program in neurodegenerative diseases, like in dying midbrain dopaminergic neurons in Parkinson's disease, is of great interest. This article reviews the recent international patents on modulation of RET kinase activity by small-molecule and peptide-based agonists and antagonists.

The structure of the glial cell line-derived neurotrophic factor-coreceptor complex: insights into RET signaling and heparin binding.

Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor alpha1 (GFR alpha 1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF(2).GFR alpha 1(2) complex structure at 2.35 A resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF(2).GFR alpha 1(2) complex and the previously published artemin(2).GFR alpha 3(2) complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFR alpha interface between the two structures, in particular ones that buttress the key conserved Arg(GFR alpha)-Glu(ligand)-Arg(GFR alpha) interaction. Second, the flexible GDNF ligand "finger" loops fit differently into the GFR alphas, which are rigid. Third, and we believe most importantly, the quaternary structure of the two tetramers is dissimilar, because the angle between the two GDNF monomers is different. This suggests that the RET-RET interaction differs in different ligand(2)-co-receptor(2)-RET(2) heterohexamer complexes. Consistent with this, we showed that GDNF(2).GFR alpha1(2) and artemin(2).GFR alpha 3(2) signal differently in a mitogen-activated protein kinase assay. Furthermore, we have shown by mutagenesis and enzyme-linked immunosorbent assays of RET phosphorylation that RET probably interacts with GFR alpha 1 residues Arg-190, Lys-194, Arg-197, Gln-198, Lys-202, Arg-257, Arg-259, Glu-323, and Asp-324 upon both domains 2 and 3. Interestingly, in our structure, sucrose octasulfate also binds to the Arg(190)-Lys(202) region in GFR alpha 1 domain 2. This may explain how GDNF.GFR alpha 1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET.

Structure of the actin-depolymerizing factor homology domain in complex with actin.

Actin dynamics provide the driving force for many cellular processes including motility and endocytosis. Among the central cytoskeletal regulators are actin-depolymerizing factor (ADF)/cofilin, which depolymerizes actin filaments, and twinfilin, which sequesters actin monomers and caps filament barbed ends. Both interact with actin through an ADF homology (ADF-H) domain, which is also found in several other actin-binding proteins. However, in the absence of an atomic structure for the ADF-H domain in complex with actin, the mechanism by which these proteins interact with actin has remained unknown. Here, we present the crystal structure of twinfilin's C-terminal ADF-H domain in complex with an actin monomer. This domain binds between actin subdomains 1 and 3 through an interface that is conserved among ADF-H domain proteins. Based on this structure, we suggest a mechanism by which ADF/cofilin and twinfilin inhibit nucleotide exchange of actin monomers and present a model for how ADF/cofilin induces filament depolymerization by weakening intrafilament interactions.

The Yersinia adhesin YadA binds to a collagenous triple-helical conformation but without sequence specificity.

The Yersinia adhesin A (YadA) is a collagen-binding trimeric autotransporter of Yersinia enterocolitica, an enteropathogen that causes a range of gastroenteric and systemic diseases, and YadA is essential for Y. enterocolitica virulence. Although previous studies suggest a specific binding site in collagen for YadA, we found that recombinant YadA binds to both major cyanogen bromide fragments of collagen type II and the collagen-like model peptide (Pro-Hyp-Gly)(10) [(POG)(10)]. To further characterise the YadA-collagen interaction, we investigated the binding of YadA to (POG)(10) and three other model peptides, (Pro-Pro-Gly)(10) which lacks the hydroxyl groups of (POG)(10), T3-785 which contains a stretch of the collagen type III sequence and Gly(-) which is similar to (POG)(10) but lacks the central glycine. All the peptides except Gly(-) adopt a collagen-like triple-helical conformation at room temperature. All three triple-helical peptides bound to YadA, with (POG)(10) being the tightest, whereas binding of Gly(-) was hardly detectable. The affinity of (POG)(10) for YadA was 0.28 microM by isothermal titration calorimetry and 0.17 microM by surface plasmon resonance (SPR), similar to that of collagen type I. Our results show that a collagen-like triple-helical conformation, strengthened by the presence of hydroxyproline residues, is both necessary and sufficient for YadA binding.