Β-propeller Domain Research Articles

Gene cloning and enzyme structure modeling of the Aspergillus oryzae N74 fructosyltransferase

The fructooligosaccharides (FOS) represent an important source of prebiotic compounds that are widely used as an ingredient in functional foods. Recently, the strain Aspergillus oryzae N74 was reported as a potential microorganism for the industrial production of FOS, due to its high yields of FOS production. In this work, we used a PCR-cloning strategy to clone the A. oryzae N74 ftase gene as a previous step for recombinant enzyme production. Ftase showed a 1630 bp size with a 99% similarity with other A. oryzae strains and between 1 to 68% identities with other Aspergillus strains. This gene encodes for a 525 amino acids protein with 99% similarity with other A. oryzae strains and between 11 to 69% similarities with other Aspergillus strains. Finally, an A. oryzae N74 FTase tertiary structure model was predicted base on its similarity with other glycoside hydrolase 32 family members. The active site was located inside the β-propeller domain and was formed for non-charged polar and charged amino acids. In summary, these results shows the high level of sequence conservation between A. oryzae strains and represent a first step towards the development of a FOS production industrial process using recombinant microorganism carrying the ftase gene from A. oryzae N74.

Pseudo-active sites of protease domains: HGF/Met and Sonic hedgehog signaling in cancer

Proteases represent a large class of enzymes with crucial biological functions. Although targeting various relevant proteases for therapeutic intervention has been widely investigated, structurally related proteins lacking proteolytic activity (pseudo-proteases) have received relatively little attention. Two distinct clinically relevant cancer pathways that contain signaling proteins with pseudo-protease domains include the Met and Hedgehog (Hh) pathways. The receptor tyrosine kinase Met pathway is driven by hepatocyte growth factor (HGF), a plasminogen-related ligand that binds Met and activates intracellular pathways resulting in cell proliferation, angiogenesis, motility and survival. HGF is a disulfide-linked alpha/beta-heterodimer having a trypsin serine protease-like beta-chain. The Hh pathway is driven by Sonic hedgehog (Shh), which has a Zn(2+) metalloprotease fold and binds Patched1 (Ptc1), which de-represses Smoothened and ultimately activates Gli-dependent transcription. Although HGF and Shh differ in structure and function, the pseudo-catalytic sites of both HGF and Shh are crucial for signal transduction. For HGF, this region binds the Met beta-propeller domain, which leads to Met dimerization and signaling. For Hh, this region binds to the antagonist receptor Hedgehog-interacting protein (Hhip) and most probably to Ptc1 as well. Thus, for both HGF and Hh pathways, targeting ligand pseudo-active sites represents a new strategy for regulation.

A unique interaction between αIIb and β3 in the head region is essential for outside-in signaling–related functions of αIIbβ3 integrin

AbstractThe main interface of the 2 subunits of platelet integrin αIIbβ3 comprises the β-propeller domain of αIIb and the βA domain of β3. In the center of the β-propeller, several aromatic residues interact by cation-π and hydrophobic bonds with Arg261 of βA. In this study, we substituted αIIb-Trp110 or β3-Arg261 by residues abundant in other α or β subunits at corresponding locations and expressed them in baby hamster kidney cells along with normal β3 or αIIb, respectively. These mutant cells displayed normal surface expression and fibrinogen binding but grossly impaired outside-in signaling–related functions: adhesion to immobilized fibrinogen, cell spreading, focal adhesion kinase phosphorylation, clot retraction, and reduced αIIbβ3 stability in EDTA (ethylenediaminetetraacetic acid). Expression of mutants with substitutions of Arg261 in β3 by alanine or lysine with normal αv yielded normal surface expression of αvβ3 and soluble fibrinogen binding as well as normal outside-in signaling–related functions, contrasting findings for αIIbβ3. Structural analysis of αIIbβ3 and αvβ3 revealed that αvβ3 has several strong interactions between αv and β3 subunits that are missing in αIIbβ3. Together, these findings indicate that the interaction between Trp110 of αIIb and Arg261 of β3 is critical for αIIbβ3 integrity and outside-in signaling–related functions.

Crystal Structure of Yeast Rpn14, a Chaperone of the 19 S Regulatory Particle of the Proteasome

The ubiquitin-proteasome pathway is a major proteolytic system in eukaryotic cells and regulates various cellular processes. The 26 S proteasome, the central enzyme of this pathway, consists of a proteolytic core particle and two 19 S regulatory particles (RPs) composed of ATPase (Rpt) and non-ATPase (Rpn) subunits. Growing evidence indicates that proteasome assembly is assisted by a variety of chaperones. In particular, it has been reported recently that Nas2, Nas6, Rpn14, and Hsm3 bind specific Rpt subunits, thereby contributing to the formation of 19 S RP. Rpn14 transiently binds to the C-terminal domain of the Rpt6 subunit (Rpt6-C) during maturation of the ATPase ring of 19 S RP. In this study, we determined the crystal structure of yeast Rpn14 at 2.0 A resolution, which revealed that this chaperone consists of a unique N-terminal domain with unknown function and a C-terminal domain assuming a canonical seven-bladed beta-propeller fold. The Rpt6-binding site on Rpn14 was predicted based on structural comparison with the complex formed between Nas6 and Rpt3-C. The top face of Rpn14 exhibits a highly acidic surface area, whereas the putative interacting surface of Rpt6-C is basic. By inspection of structural data along with genetic and biochemical data, we determined the specific residues of Rpn14 and Rpt6 for complementary charge interactions that are required for 19 S RP assembly.

Dimeric architecture of the Hendra virus attachment glycoprotein: evidence for a conserved mode of assembly.

Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed beta-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to alpha-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60 degrees ) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses.

Mechanism of LDL binding and release probed by structure-based mutagenesis of the LDL receptor

The LDL receptor (LDL-R) mediates cholesterol metabolism in humans by binding and internalizing cholesterol transported by LDL. Several different molecular mechanisms have been proposed for the binding of LDL to LDL-R at neutral plasma pH and for its release at acidic endosomal pH. The crystal structure of LDL-R at acidic pH shows that the receptor folds back on itself in a closed form, obscuring parts of the ligand binding domain with the epidermal growth factor (EGF)-precursor homology domain. We have used a structure-based site-directed mutagenesis approach to examine 12 residues in the extracellular domain of LDL-R for their effect on LDL binding and release. Our studies show that the interface between the ligand binding domain and the EGF-precursor homology domain seen at acidic pH buries residues mediating both LDL binding and release. Our results are consistent with an alternative model of LDL-R whereby multiple modules of the extracellular domain interact with LDL at neutral pH, concurrently positioning key residues so that at acidic pH the LDL-R:LDL interactions become unfavorable, triggering release. After LDL release, the closed form of LDL-R may target its return to the cell surface.

Keap1 is a forked-stem dimer structure with two large spheres enclosing the intervening, double glycine repeat, and C-terminal domains

Keap1 is a substrate adaptor of a Cullin 3-based E3 ubiquitin ligase complex that recognizes Nrf2, and also acts as a cellular sensor for xenobiotics and oxidative stresses. Nrf2 is a transcriptional factor regulating the expression of cytoprotective enzyme genes in response to such stresses. Under unstressed conditions Keap1 binds Nrf2 and results in rapid degradation of Nrf2 through the proteasome pathway. In contrast, upon exposure to oxidative and electrophilic stress, reactive cysteine residues in intervening region (IVR) and Broad complex, Tramtrack, and Bric-à-Brac domains of Keap1 are modified by electrophiles. This modification prevents Nrf2 from rapid degradation and induces Nrf2 activity by repression of Keap1. Here we report the structure of mouse Keap1 homodimer by single particle electron microscopy. Three-dimensional reconstruction at 24-A resolution revealed two large spheres attached by short linker arms to the sides of a small forked-stem structure, resembling a cherry-bob. Each sphere has a tunnel corresponding to the central hole of the beta-propeller domain, as determined by x-ray crystallography. The IVR domain appears to surround the core of the beta-propeller domain. The unexpected proximity of IVR to the beta-propeller domain suggests that any distortions generated during modification of reactive cysteine residues in the IVR domain may send a derepression signal to the beta-propeller domain and thereby stabilize Nrf2. This study thus provides a structural basis for the two-site binding and hinge-latch model of stress sensing by the Nrf2-Keap1 system.

The crystal structure of the TolB box of colicin A in complex with TolB reveals important differences in the recruitment of the common TolB translocation portal used by group A colicins

Interaction of the TolB box of Group A colicins with the TolB protein in the periplasm of Escherichia coli cells promotes transport of the cytotoxic domain of the colicin across the cell envelope. The crystal structure of a complex between a 107-residue peptide (TA1–107) of the translocation domain of colicin A (ColA) and TolB identified the TolB box as a 12-residue peptide that folded into a distorted hairpin within a central canyon of the β-propeller domain of TolB. Comparison of this structure with that of the colicin E9 (ColE9) TolB box–TolB complex, together with site-directed mutagenesis of the ColA TolB box residues, revealed important differences in the interaction of the two TolB boxes with an overlapping binding site on TolB. Substitution of the TolB box residues of ColA with those of ColE9 conferred the ability to competitively recruit TolB from Pal but reduced the biological activity of the mutant ColA. This datum explains (i) the difference in binding affinities of ColA and ColE9 with TolB, and (ii) the inability of ColA, unlike ColE9, to competitively recruit TolB from Pal, allowing an understanding of how these two colicins interact in a different way with a common translocation portal in E. coli cells.

Reconstitution of a Frizzled8·Wnt3a·LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6

Wnt/beta-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/beta-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD.Wnt3a.LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth beta-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/beta-catenin signaling by Dkk1.

Computational Prediction and Experimental Validation of a Novel Binding Site for Platelet Integrin AlphaIIb-Beta3

Fibrinogen mimetic drugs and a γ-chain peptide bind to both the αIIb and β3 subunits of integrin αIIbβ3, providing the final coordination site for the metal ion-dependent adhesion site (MIDAS) metal ion within the β3 I domain, and inducing a less compact conformation of the protein. We investigated the binding of a novel human-selective αIIbβ3 small molecular inhibitor (RUC-1) that we recently identified by high throughput screening, and predicted to bind preferentially to a pocket within the αIIb β-propeller domain by flexible ligand/rigid protein molecular docking studies. We first carried out both standard and enhanced molecular dynamics (MD) simulations of the proposed RUC-1-bound form of human αIIbβ3 integrin. The results of these studies pointed to an energetically preferred conformation of RUC-1 into the αIIb β-propeller domain that had no contact with the MIDAS metal ion or other sites in the β3 I domain. This binding mode of RUC-1 appeared to be stabilized by interactions with specific human αIIb residues, such as D224 (already known to contribute to αIIbβ3 binding), Y190 (F in both mouse and rat), and (through two water molecules) D232 (H in rat). Well-tempered metadynamics simulations of Y190F and D232H mutants supported the contribution of normal residues to the stabilization of RUC-1 in a specific binding mode and location. Functional experiments on recombinant cell lines expressing Y190FαIIbβ3 or D232HαIIbβ3 validated this hypothesis by showing a ∼80-95% reduction in RUC-1 affinity. X-ray crystallography confirmed the RUC-1 binding pose suggested by MD simulations, while gel filtration and dynamic light scattering experiments showed that RUC-1 favored a compact αIIbβ3 conformation, in sharp contrast to the effect of fibrinogen-mimetic drugs.

Structural, Functional, and Dynamic Characterization of the Binding Site of RUC-1, a Novel αIIb-Specific Inhibitor of Integrin αIIbβ3.

Abstract 151The platelet integrin αIIbβ3 mediates platelet aggregation by binding the adhesive plasma proteins fibrinogen and von Willebrand factor. We previously reported that co-crystallizing the αIIbβ3 headpiece with either of the two ligand mimetic drugs eptifibatide or tirofiban, or with a fibrinogen γ-chain peptide, results in the αIIbβ3 headpiece adopting an open conformation characterized by a swing-out motion of the β3 hybrid domain. In addition, all of the compounds bound both to the αIIb and β3 subunits, with the β3 contact mediated in part by coordinating the MIDAS metal ion. We recently reported on RUC-1, a novel αIIbβ3-specific small molecular antagonist that we predicted from molecular docking and biochemical data bound exclusively to αIIb. We have now determined the crystal structure of the αIIbβ3 headpiece in the absence of ligand and after soaking in RUC-1, and correlated the findings with advanced molecular dynamics simulations and functional studies of mutant receptors to define the mechanism of RUC-1 binding. The crystal structure shows that the αaIIbβ3 headpiece in the absence of RUC-1 or other ligand adopts a closed conformation with minimal β3 swing-out, similar to what we found in the full-length ectodomain of the bent receptor in the absence of ligand. Strikingly, RUC-1 only binds to the pocket formed by the αIIb β-propeller domain and has no contact with MIDAS metal ion or other sites in the β3 I domain. Standard molecular dynamics studies revealed a very similar binding pocket for RUC-1, which reflects the lowest energy binding state as assessed by metadynamics simulations. Both the crystal structure and the metadynamics studies identified αIIb Y190 and αIIb D232 as important for RUC-1 binding. RUC-1 is a much less potent inhibitor of mouse and rat αIIbβ3 than human αIIbβ; both mouse and rat have αIIb Y190F substitutions, and in addition, rat αIIb has a D232H substitution. A recombinant cell line expressing Y190F αIIbβ3 demonstrated an ∼80% reduction in RUC-1 affinity as judged by a 6-fold increase in IC50 for inhibiting fibrinogen binding and one expressing D232H αIIb β3 had more than a 95% decrease in affinity for RUC-1. To test whether RUC-1's exclusive binding to the αIIb subunit was associated with a reduced ability to induce a conformational change in αIIbβ3, we performed gel filtration and dynamic light scattering studies. In sharp contrast to the effects of eptifibatide and tirofiban, RUC-1 did not induce αIIbβ3 to adopt a less compact conformation in either system. These data suggest that ligand engagement of the β3 I domain, in particular coordinating the MIDAS metal ion and stabilizing the β1-α1 loop movement toward the MIDAS, is a likely trigger for β3 undergoing its swing-out motion. These data have potential therapeutic implications because they raise the possibility of inhibiting αIIbβ3 with an αIIb-specific antagonist that does not initiate the conformational changes in β3 that have been hypothesized to underlie the development of two major toxicities of αIIbβ3 antagonists, thrombocytopenia and paradoxical activation of the receptor. Disclosures:Filizola:Mount Sinai School of Medicine: Employment, Patents & Royalties. Coller:Rockefeller University: Employment, Patents & Royalties; Research Foundation of the State University of New York: Patents & Royalties; Centocor: Patents & Royalties; Accumetrics: Consultancy, Patents & Royalties.

Mapping the ligand-binding pocket of integrin α5β1 using a gain-of-function approach

Integrin alpha5beta1 is a key receptor for the extracellular matrix protein fibronectin. Antagonists of human integrin alpha5beta1 have therapeutic potential as anti-angiogenic agents in cancer and diseases of the eye. However, the structure of the integrin is unsolved and the atomic basis of fibronectin and antagonist binding by integrin alpha5beta1 is poorly understood. In the present study, we demonstrate that zebrafish alpha5beta1 integrins do not interact with human fibronectin or the human alpha5beta1 antagonists JSM6427 and cyclic peptide CRRETAWAC. Zebrafish alpha5beta1 integrins do bind zebrafish fibronectin-1, and mutagenesis of residues on the upper surface and side of the zebrafish alpha5 subunit beta-propeller domain shows that these residues are important for the recognition of the Arg-Gly-Asp (RGD) motif and the synergy sequence [Pro-His-Ser-Arg-Asn (PHSRN)] in fibronectin. Using a gain-of-function analysis involving swapping regions of the zebrafish integrin alpha5 subunit with the corresponding regions of human alpha5 we show that blades 1-4 of the beta-propeller are required for human fibronectin recognition, suggesting that fibronectin binding involves a broad interface on the side and upper face of the beta-propeller domain. We find that the loop connecting blades 2 and 3 of the beta-propeller, the D3-A3 loop, contains residues critical for antagonist recognition, with a minor role played by residues in neighbouring loops. A new homology model of human integrin alpha5beta1 supports an important function for D3-A3 loop residues Trp157 and Ala158 in the binding of antagonists. These results will aid the development of reagents that block integrin alpha5beta1 functions in vivo.

Low pH-Triggered Beta-Propeller Switch of the Low-Density Lipoprotein Receptor Assists Rhinovirus Infection

Minor group human rhinoviruses (HRVs) bind three members of the low-density lipoprotein receptor (LDLR) family: LDLR proper, very-LDLR (VLDLR) and LDLR-related protein (LRP). Whereas ICAM-1, the receptor of major group HRVs actively contributes to viral uncoating, LDLRs are rather considered passive vehicles for cargo delivery to the low-pH environment of endosomes. Since the Tyr-Trp-Thr-Asp beta-propeller domain of LDLR has been shown to be involved in the dissociation of bound LDL via intramolecular competition at low pH, we studied whether it also plays a role in HRV infection. Human cell lines deficient in LDLR family proteins are not available. Therefore, we used CHO-ldla7 cells that lack endogenous LDLR. These were stably transfected to express either wild-type (wt) human LDLR or a mutant with a deletion of the beta-propeller. When HRV2 was attached to the propeller-negative LDLR, a lower pH was required for conversion to subviral particles than when attached to wt LDLR. This indicates that high-avidity receptor binding maintains the virus in its native conformation. HRV2 internalization directed the mutant LDLR but not wt LDLR to lysosomes, resulting in reduced plasma membrane expression of propeller-negative LDLR. Infection assays using a CHO-adapted HRV2 variant showed a delay in intracellular viral conversion and de novo viral synthesis in cells expressing the truncated LDLR. Our data indicate that the beta-propeller attenuates the virus-stabilizing effect of LDLR binding and thereby facilitates RNA release from endosomes, resulting in the enhancement of infection. This is a nice example of a virus exploiting high-avidity multimodule receptor binding with an intrinsic release mechanism.

Molecular Interplay between Endostatin, Integrins, and Heparan Sulfate

Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. alpha5beta1 and alphavbeta3 integrins form stable complexes with immobilized endostatin (KD=approximately 1.8x10(-8) M, two-state model). Two arginine residues (Arg27 and Arg139) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the alphavbeta3 integrin at the interface between the beta-propeller domain of the alphav subunit and the betaA domain of the beta3 subunit. In addition, we report that alpha5beta1 and alphavbeta3 integrins bind to heparin/heparan sulfate. The ectodomain of the alpha5beta1 integrin binds to haparin with high affinity (KD=15.5 nM). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and alpha5beta1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.

A PH domain within OCRL bridges clathrin-mediated membrane trafficking to phosphoinositide metabolism

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5-phosphatase domain followed by an ASH and a RhoGAP-like domain. Their divergent NH2-terminal portions remain uncharacterized. We show that the NH2-terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin-coated pits, was earlier shown to contain another binding site for clathrin in its COOH-terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2-terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin-binding site in OCRL maps to an unusual clathrin-box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin-dependent membrane trafficking.

Cooperative Folding and Ligand-binding Properties of LRP6 β-Propeller Domains

Wnt/beta-catenin signaling controls cell growth during development, and its misregulation in adults can cause human diseases. LRP6, the essential co-receptor for the Wnt pathway, consists of four beta-propeller domains flanked by epidermal growth factor repeats in its extracellular region. To understand the maturation and ligand-binding properties of individual BP domains, we generated soluble receptor consisting of individual BPs, as well as combinations of these domains. We show that BP1, BP2, and BP4 each can be folded and secreted, and their secretion was enhanced by co-expression of Mesd, a molecular chaperone essential for LRP6 folding and maturation. BP3 is not secreted when expressed on its own or in combination with BP2 or BP1 and 2 (BP12); however, folding and secretion of BP3 is vastly enhanced when expressed together with BP4. Similar cooperative folding and maturation was observed between BP1 and BP2. These results suggest that BP1 forms a functional folding unit with BP2, whereas BP3 folds together with BP4. Using these BP constructs, we also found that BP12 and BP34 constitute independent ligand-binding domains capable of binding Wnt3a, Dkk1, and Mesd. The ability of Mesd to block the binding of both Wnt3a and Dkk1 to LRP6 enables this specialized chaperone to function as a Wnt signaling modulator. Together, our studies reveal unique properties of the LRP6 BP domains and provide novel tools to understand LRP6 function in ligand binding and Wnt signaling. Our results also support the development of soluble LRP6 receptors and Mesd as potential therapeutic molecules that target Wnt signaling.

Conformation Coupling Between The I-like Domain Alpha7 Helix And The Hybrid Domain Of Beta3 Integrins

Integrins are αβ heterodimers that mediate cell adhesion and transduce signals bidirectionally across the cell membrane. Integrins often exist in low affinity (or inactive) states for ligand binding on the cell curface, but change conformations to high affinity (or activated) states when induced by stimuli from inside or outside the cell. Crystallographic and electron microscopic studies have obtained evidence that the low affinity states correspond to bent conformations where the integrin headpiece interacts with the legs, whereas the high affinity states correspond to more extended conformations where integrins stand up, suggesting a switchblade-like unbending model for integrin activation. In this model, the key conformational changes on integrin headpiece include the swing out of the hybrid domain and the downward movement of I-like domain α7 helix. These conformational changes are suggested by crystal structures of β3 integrin headpieces. The unliganded αvβ3 headpiece displays a closed hybrid domain with the I-like α7 helix in an upper position and the pseudo-liganded αIIbβ3 headpiece shows an open hybrid domain with the I-like α7 helix in a lower position. Using molecular dynamics simulations we studied the stability of the two conformations of the I-like domain α7 helix and the hybrid domain, the transition between the two conformations in each structure, and the coupling between the conformational changes in the two structures. We observed that the β-propeller domain of the α subunit helped stabilize the hybrid domain at the close conformation. The down/upward movements of the I-like α7 helix correlated with the opening/closing of the hybrid domain. Our simulations identify key residues to the α7 helix movement and suggest the existence of intermediate conformations of the hybrid domain between the open and the close conformations.

Ligands bind to Sortilin in the tunnel of a ten-bladed β-propeller domain

The structure of the Sortilin ectodomain in complex with neurotensin has been determined at 2-A resolution, revealing that the C-terminal part of neurotensin binds in the tunnel of a ten-bladed beta-propeller domain. Binding competition studies suggest that additional binding sites, for example, for the prodomain of nerve growth factor-beta, are present in the tunnel and that competition for binding relates to the restricted space inside the propeller.

A structural and biochemical basis for PAPS-independent sulfuryl transfer by aryl sulfotransferase from uropathogenic Escherichia coli

Sulfotransferases are a versatile class of enzymes involved in numerous physiological processes. In mammals, adenosine 3'-phosphate-5'-phosphosulfate (PAPS) is the universal sulfuryl donor, and PAPS-dependent sulfurylation of small molecules, including hormones, sugars, and antibiotics, is a critical step in hepatic detoxification and extracellular signaling. In contrast, little is known about sulfotransferases in bacteria, which make use of sulfurylated molecules as mediators of cell-cell interactions and host-pathogen interactions. Bacterial arylsulfate sulfotransferases (also termed aryl sulfotransferases), in contrast to PAPS-dependent sulfotransferases, transfer sulfuryl groups exclusively among phenolic compounds in a PAPS-independent manner. Here, we report the crystal structure of the virulence factor arylsulfate sulfotransferase (ASST) from the prototypic, pyelonephritogenic Escherichia coli strain CFT073 at 2.0-A resolution, and 2 catalytic intermediates, at 2.1-A and 2.4-A resolution, with substrates bound in the active site. ASST is one of the largest periplasmic enzymes and its 3D structure differs fundamentally from all other structurally characterized sulfotransferases. Each 63.8-kDa subunit of the ASST homodimer comprises a 6-bladed beta-propeller domain and a C-terminal beta-sandwich domain. The active sites of the dimer are situated at the center of the channel formed by each beta-propeller and are defined by the side chains of His-252, His-356, Arg-374, and His-436. We show that ASST follows a ping-pong bi-bi reaction mechanism, in which the catalytic residue His-436 undergoes transient sulfurylation, a previously unreported covalent protein modification. The data provide a framework for understanding PAPS-independent sulfotransfer and a basis for drug design targeting this bacterial virulence factor.

Genetically Determined Proteolytic Cleavage Modulates α7β1 Integrin Function

The dystrophin-glycoprotein complex and the alpha7beta1 integrin are trans-sarcolemmal linkage systems that connect and transduce contractile forces between muscle fibers and the extracellular matrix. alpha7beta1 is the major laminin binding integrin in skeletal muscle. Different functional variants of this integrin are generated by alternative splicing and post-translational modifications such as glycosylation and ADP-ribosylation. Here we report a species-specific difference in alpha7 chains that results from an intra-peptide proteolytic cleavage, by a serine protease, at the 603RRQ605 site. Site-directed mutagenesis of RRQ to GRQ prevents this cleavage. This RRQ sequence in the alpha7 integrin chain is highly conserved among vertebrates but it is absent in mice. Protein structure modeling indicates this cleavage site is located in an open region between the beta-propeller and thigh domains of the alpha7 chain. Compared with the non-cleavable alpha7 chain, the cleaved form enhances cell adhesion and spreading on laminin. Cleavage of the alpha7 chain is elevated upon myogenic differentiation, and this cleavage may be mediated by urokinase-type plasminogen activator. These results suggest proteolytic cleavage is a novel mechanism that regulates alpha7 integrin functions in skeletal muscle, and that the generation of such cleavage sites is another evolutionary mechanism for expanding and modifying protein functions.