Structural determinants of RNA recognition and cleavage by Dicer

A hallmark of RNA interference is the production of short double-stranded RNA (dsRNA) molecules 21-28 nucleotides in length by the specialized RNase III protein Dicer. Dicer enzymes uniquely generate RNA products of specific lengths by mechanisms that have not been fully elucidated. Here we show that the PAZ domain responsible for dsRNA end recognition confers this measuring ability through both its structural position and RNA-binding specificity. Point mutations define the dsRNA-binding surface and reveal a protein loop important for cleavage of substrates containing perfect or imperfect base pairing. On the basis of these results, we reengineered Dicer with a U1A RNA-binding domain in place of the PAZ domain to create an enzyme with altered end-recognition specificity and RNA product length. These results explain how Dicer functions as a molecular ruler and provide a structural basis for modifying its activity in cells.     

Structural determinants of integrin recognition by talin

The binding of cytoplasmic proteins, such as talin, to the cytoplasmic domains of integrin adhesion receptors mediates bidirectional signal transduction. Here we report the crystal structure of the principal integrin binding and activating fragment of talin, alone and in complex with fragments of the beta 3 integrin tail. The FERM (four point one, ezrin, radixin, and moesin) domain of talin engages integrins via a novel variant of the canonical phosphotyrosine binding (PTB) domain-NPxY ligand interaction that may be a prototype for FERM domain recognition of transmembrane receptors. In combination with NMR and mutational analysis, our studies reveal the critical interacting elements of both talin and the integrin beta 3 tail, providing structural paradigms for integrin linkage to the cell interior.     

Structural and dynamic determinants of protein-peptide recognition

Protein-peptide interactions play important roles in many cellular processes, including signal transduction, trafficking, and immune recognition. Protein conformational changes upon binding, an ill-defined peptide binding surface, and the large number of peptide degrees of freedom make the prediction of protein-peptide interactions particularly challenging. To address these challenges, we perform rapid molecular dynamics simulations in order to examine the energetic and dynamic aspects of protein-peptide binding. We find that, in most cases, we recapitulate the native binding sites and native-like poses of protein-peptide complexes. Inclusion of electrostatic interactions in simulations significantly improves the prediction accuracy. Our results also highlight the importance of protein conformational flexibility, especially side-chain movement, which allows the peptide to optimize its conformation. Our findings not only demonstrate the importance of sufficient sampling of the protein and peptide conformations, but also reveal the possible effects of electrostatics and conformational flexibility on peptide recognition.     

Identification of novel matrix metalloproteinase-7 (matrilysin) cleavage sites in murine and human Fas ligand

Soluble Fas ligand (sFasL) is released from the cell surface by matrix metalloproteinases (MMPs), one of which is MMP-7. We have reported that MMP-7-generated sFasL is pro-apoptotic in both in vitro and in vivo systems. However, there are contradictory reports that the soluble form of FasL is inactive or anti-apoptotic, resulting in significant controversy in the literature. One potential explanation for these discrepancies is that forms of sFasL with different amino-terminal sequences have been demonstrated to have varying activities. Here we report that MMP-7 cleaves murine and human FasL at sites that are distinct from previously reported cleavage sites resulting in production of novel forms of sFasL. Cleavage of FasL by MMP-7 occurs at the leucine residues in the sequence "ELAELR" within the region between the transmembrane and trimerization domains. When this site is unavailable, a more c-terminal site, "SL," is cleaved. MMP-7 differentially processes murine and human FasL since it cleaves human FasL not only at the "ELAELR" site but also at a previously identified site. Additionally, MMP-3, but not MMP-2, was found to have the same cleavage specificity for murine FasL as MMP-7. We conclude that the controversy regarding the biological activity of sFasL may be explained, in part, by the generation of distinct forms of sFasL as a result of cleavage at specific sequences.     

Structural determinants of sphingolipid recognition by commercially available anti-ceramide antibodies

The sphingolipid mediator ceramide is involved in cellular processes such as apoptosis, differentiation, responses to cytokines, and stress responses. Experimental evidence suggests that the intracellular location of ceramide may be a key factor in determining its ultimate cellular effects. One approach to ceramide localization is the use of recently developed anti-ceramide antibodies for immunocytochemical studies. Two such commercial preparations are now available; we sought to compare and contrast their specificity for ceramide and/or other cellular lipids. By using lipid overlay assays and a diverse panel of sphingolipids, we were able to delineate the specificity and thus, the utility of these reagents. Our results indicate that one of these anti-ceramide preparations is quite specific for ceramide and dihydroceramide, whereas the other preparation recognizes dihydroceramide, phosphatidylcholine, and sphingomyelin. Furthermore, through the use of chemically modified ceramides in similar assays, we were able to determine some structural determinants of lipid recognition by both of these reagents.     

Structural basis of coagulation factor V recognition for cleavage by RVV-V

Russell's viper venom factor V (FV) activator (RVV-V) is a thrombin-like proteinase that specifically cleaves the Arg1545-Ser1546 bond of FV. Here we present the crystal structure of RVV-V in complex with the FV14 peptide (residues 1533-1546 of human FV) determined at 1.8? resolution. The structure reveals multiple interactions between RVV-V and the seven residues, Ile1539 (P(7))-Arg1545 (P(1)), of the cleaved substrate. Comparison with substrate-free structures reveals conformational changes of the RVV-V loops upon substrate binding, suggesting that the multiple interactions are mediated by an induced-fit mechanism. The results provide an explanation for the narrow specificity of RVV-V.     

Structural determinants of SecB recognition by SecA in bacterial protein translocation

SecB is a bacterial chaperone involved in directing pre-protein to the translocation pathway by its specific interaction with the peripheral membrane ATPase SecA. The SecB-binding site on SecA is located at its C terminus and consists of a stretch of highly conserved residues. The crystal structure of SecB in complex with the C-terminal 27 amino acids of SecA from Haemophilus influenzae shows that the SecA peptide is structured as a CCCH zinc-binding motif. One SecB tetramer is bound by two SecA peptides, and the interface involves primarily salt bridges and hydrogen bonding interactions. The structure explains the importance of the zinc-binding motif and conserved residues at the C terminus of SecA in its high-affinity binding with SecB. It also suggests a model of SecB-SecA interaction and its implication for the mechanism of pre-protein transfer in bacterial protein translocation.     

Structural determinants of a glucocorticoid receptor recognition element

Analysis of the relative inducibility of an extensive series of mutant glucocorticoid response elements (GREs) defines features critical to the constitution of an active GRE. Assuming that function as a GRE reflects binding of glucocorticoid receptor, our activity data are consistent with the recognition of the GRE as two hexamer half-sites, each half-site recognized by a single subunit of a receptor dimer, probably in a cooperative fashion. Integrity of both half-sites is necessary for an active element, and spacing of the half-sites is critical. The identity of 1 basepair within the hexamer half-site is unconstrained; the receptor probably makes no base-specific contacts at this position. In contrast, at other positions within the half-site, limited substitutions (if any) can be tolerated. These results along with data from certain insertion mutations suggest that the receptor recognizes each hexamer half-site as two separable subelements. A further implication is that the DNA-binding domain of the glucocorticoid receptor is composed of distinct subdomains, which interact with the subelements of the recognition sequence.     

Structural basis of pre-mRNA recognition by the human cleavage factor Im complex

The cleavage factor I(m) (CF I(m)), consists of a 25 kDa subunit (CF I(m)25) and one of three larger subunits (CF I(m)59, CF I(m)68, CF I(m)72), and is an essential protein complex for pre-mRNA 3'-end cleavage and polyadenylation. It recognizes the upstream sequence of the poly(A) site in a sequence-dependent manner. Here we report the crystal structure of human CF I(m), comprising CF I(m)25 and the RNA recognition motif domain of CF I(m)68 (CF I(m)68RRM), and the crystal structure of the CF I(m)-RNA complex. These structures show that two CF I(m)68RRM molecules bind to the CF I(m)25 dimer via a novel RRM-protein interaction mode forming a heterotetramer. The RNA-bound structure shows that two UGUAA RNA sequences, with anti-parallel orientation, bind to one CF I(m)25-CF I(m)68RRM heterotetramer, providing structural basis for the mechanism by which CF I(m) binds two UGUAA elements within one molecule of pre-mRNA simultaneously. Point mutation and kinetic analyses demonstrate that CF I(m)68RRM can bind the immediately flanking upstream region of the UGUAA element, and CF I(m)68RRM binding significantly increases the RNA-binding affinity of the complex, suggesting that CF I(m)68 makes an essential contribution to pre-mRNA binding.     

Cleavage of neuronal synaptosomal-associated protein of 25 kDa by exogenous matrix metalloproteinase-7

Matrix metalloproteinases (MMPs) belong to a family of zinc dependent enzymes best studied for their role in cancer and inflammation. Though MMPs typically target extracellular proteins, here we show that MMP-7, an MMP family member which lacks a C-terminal hemopexin-like domain, can cleave an intraneuronal protein that is critical to vesicular fusion and neurotransmitter release, synaptosomal-associated protein of 25 kDa (SNAP-25). Western blot analysis using an N-terminal specific antibody on extracts from cultured neurons suggests that cleavage occurs towards the C-terminal portion of SNAP 25. Additional studies with recombinant SNAP-25 demonstrate that cleavage occurs at amino acid 129. The ability of MMP-7 to cleave SNAP-25 is diminished by chlorpromazine and phenylarsine oxide, inhibitors of clathrin dependent endocytosis. Together, these results imply that exogenous MMP-7 can access an intraneuronal substrate and suggest that additional studies may be warranted to determine if SNAP function is impaired with brain inflammation.     

A unique substrate recognition profile for matrix metalloproteinase-2.

The catalytic domains of the matrix metalloproteinases (MMPs) are structurally homologous, raising questions as to the degree of distinction, or overlap, in substrate recognition. The primary objective of the present study was to define the substrate recognition profile of MMP-2, a protease that was historically referred to as gelatinase A. By cleaving a phage peptide library with recombinant MMP-2, four distinct sets of substrates were identified. The first set is structurally related to substrates previously reported for other MMPs. These substrates contain the PXX/X(Hy) consensus motif (where X(Hy) is a hydrophobic residue) and are not generally selective for MMP-2 over the other MMPs tested. Two other groups of substrates were selected from the phage library with similar frequency. Substrates in group II contain the L/IXX/X(Hy) consensus motif. Substrates in group III contain a consensus motif with a sequence of X(Hy)SX/L, and the fourth set of substrates contain the HXX/X(Hy) sequence. Substrates in Group II, III, and IV were found to be 8- to almost 200-fold more selective for MMP-2 over MMP-9. To gain an understanding of the structural basis for substrate selectivity, individual residues within substrates were mutated, revealing that the P(2) residue is a key element in conferring selectivity. These findings indicate that MMP-2 and MMP-9 exhibit different substrate recognition profiles and point to the P(2) subsite as a primary determinant in substrate distinction.     

Binding,selectivity and sequence recognition of matrix metalloproteinase-2 to oligopeptides

Communicated by Ramaswamy H. Sarma     

Structural determinants of herpesvirus entry mediator recognition by murine B and T lymphocyte attenuator

The B and T lymphocyte attenuator (BTLA) appears to act as a negative regulator of T cell activation and growth. BTLA specifically interacts with herpesvirus entry mediator (HVEM), a member of the TNFR family. Herein, we have undertaken surface plasmon resonance studies to quantitatively assess BTLA and HVEM ectodomain interactions. We find that soluble BALB/cJ BTLA engages HVEM with an equilibrium affinity of 0.97+/-0.19 microM while the C57BL/6 BTLA binds slightly better with an equilibrium affinity of 0.42+/-0.06 microM. Despite its lower affinity for HVEM, the kinetic half-life of BALB/cJ BTLA complexes are twice as long as observed for C57BL/6 BTLA (4 vs 2 s). To further explore these interactions, we solved the crystal structure of a murine BTLA (BALB/cJ) ectodomain at 1.8-A resolution, revealing a beta sandwich fold with strong similarity to I-set members of the Ig superfamily. Using a structure-based mutagenesis strategy, we then examined the individual contributions of 26 BTLA surface-exposed residues toward HVEM binding. Four single-site substitutions were identified that decrease HVEM binding below detectable levels and two that decrease binding by more than half. All six of these cluster at the edge of the beta sandwich in a membrane distal patch formed primarily from the A and G strands. This patch falls within the contacting surface recently revealed in the crystal structure of the human BTLA-HVEM cocomplex. The critical binding residues identified here are highly conserved across species, suggesting that BTLA employs a conserved binding mode for HVEM recognition.     

Matrix metalloproteinase-1 cleavage site recognition and binding in full-length human type III collagen

Matrix metalloproteinases (MMPs) are essential for normal collagen turnover, recovery from fibrosis, and vascular permeability. In fibrillar collagens, MMP-1, MMP-8, and MMP-13 cleave a specific glycine–isoleucine or glycine–leucine bond, despite the presence of this sequence in other parts of the protein. This cut site specificity has been hypothesized to arise from a unique, relaxed super-secondary structure in this area due to local hydroxyproline poor character. In this study we examined the mechanism of interaction and cleavage of human type III collagen by fibroblast MMP-1 by using a panel of recombinant human type III collagens (rhCIIIs) containing engineered sequences in the vicinity of the cleavage site. Native and recombinant type III collagens had similar biochemical and structural characteristics, as indicated by transmission electron microscopy, circular dichroism spectropolarimetry, melting temperature and hydroxyproline analysis. A single amino acid change at the I785 cleavage site to proline resulted in partial MMP-1 resistance, but cuts were found in novel sites in the original cleavage region. However, the replacement of five Y-position residues by proline in this region, regardless of I785 variation, conferred complete resistance to MMP-1, MMP-8, MMP-13, trypsin, and elastase. MMP-1 had a decreased specific activity towards and reduced cleavage rate of rhCIII I785P but a K_m similar to wild-type. Despite the reductions in protease sensitivity, MMP-1 bound to all of the engineered rhCIIIs with comparable affinity, indicating that MMP-1 binding is not sufficient for cleavage. The relaxed tertiary structure in the MMP cleavage region may permit local collagen unwinding by MMP-1 that enables site-specific proteolysis.     

Osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin)

Osteopontin (OPN) is a secreted phosphoprotein shown to function in wound healing, inflammation, and tumor progression. Expression of OPN is often co-localized with members of the matrix metalloproteinase (MMP) family. We report that OPN is a novel substrate for two MMPs, MMP-3 (stromelysin-1) and MMP-7 (matrilysin). Three cleavage sites were identified for MMP-3 in human OPN, and two of those sites were also cleaved by MMP-7. These include hydrolysis of the human Gly166-Leu167, Ala201-Tyr202 (MMP-3 only), and Asp210-Leu211 peptide bonds. Only the N-terminal Gly-Leu cleavage site is conserved in rat OPN (Gly151-Leu152). These sites are distinct from previously reported cleavage sites in OPN for the proteases thrombin or enterokinase. We found evidence for the predicted MMP cleavage fragments of OPN in vitro in tumor cell lines, and in vivo in remodeling tissues such as the postpartum uterus, where OPN and MMPs are co-expressed. Furthermore, cleavage of OPN by MMP-3 or MMP-7 potentiated the function of OPN as an adhesive and migratory stimulus in vitro through cell surface integrins. We predict that interaction of MMPs with OPN at tumor and wound healing sites in vivo may be a mechanism of regulation of OPN bioactivity.     

Structural requirements for bone sialoprotein binding and modulation of matrix metalloproteinase-2

Bone sialoprotein (BSP) has been shown to induce limited gelatinase activity in latent matrix metalloproteinase-2 (MMP-2) without removal of the propeptide and to restore enzymatic activity to MMP-2 previously inhibited by tissue inhibitor of matrix metalloproteinase-2 (TIMP2). The current study identifies structural domains in human BSP and MMP-2 that contribute to these interactions. The 26 amino acid domain encoded by exon 4 of BSP is shown by a series of binding and activity assays to be involved in the displacement of MMP-2's propeptide from the active site and thereby inducing the protease activity. Binding assays in conjunction with enzyme activity assays demonstrate that both amino- and carboxy-terminal domains of BSP contribute to restoration of activity to TIMP2-inhibited MMP-2, while the MMP-2 hemopexin domain is not required for reactivation.     

Substrate hydrolysis by matrix metalloproteinase-9

The catalytic clefts of all matrix metalloproteinases (MMPs) have a similar architecture, raising questions about the redundancy in substrate recognition across the protein family. In the present study, an unbiased phage display strategy was applied to define the substrate recognition profile of MMP-9. Three groups of substrates were identified, each occupying a distinct set of subsites within the catalytic pocket. The most prevalent motif contains the sequence Pro-X-X-Hy-(Ser/Thr) at P(3) through P(2'). This sequence is similar to the MMP cleavage sites within the collagens and is homologous to substrates the have been selected for other MMPs. Despite this similarity, most of the substrates identified here are selective for MMP-9 over MMP-7 and MMP-13. This observation indicates that substrate selectivity is conferred by key subsite interactions at positions other than P(3) and P(1'). This study shows that MMP-9 has a unique preference for Arg at both P(2) and P(1), and a preference for Ser/Thr at P(2'). Substrates containing the consensus MMP-9 recognition motif were used to query the protein data bases. A surprisingly limited list of putative physiologic substrates was identified. The functional implications of these proteins lead to testable hypotheses regarding physiologic substrates for MMP-9.     

Matrilysin/matrix metalloproteinase-7(MMP7) cleavage of perlecan/HSPG2 creates a molecular switch to alter prostate cancer cell behavior

Perlecan/HSPG2, a large heparan sulfate (HS) proteoglycan, normally is expressed in the basement membrane (BM) underlying epithelial and endothelial cells. During prostate cancer (PCa) cell invasion, a variety of proteolytic enzymes are expressed that digest BM components including perlecan. An enzyme upregulated in invasive PCa cells, matrilysin/matrix metalloproteinase-7 (MMP-7), was examined as a candidate for perlecan proteolysis both in silico and in vitro. Purified perlecan showed high sensitivity to MMP-7 digestion even when fully decorated with HS or when presented in native context connected with other BM proteins. In both conditions, MMP-7 produced discrete perlecan fragments corresponding to an origin in immunoglobulin (Ig) repeat region domain IV. While not predicted by in silico analysis, MMP-7 cleaved every subpart of recombinantly generated perlecan domain IV. Other enzymes relevant to PCa that were tested had limited ability to cleave perlecan including prostate specific antigen, hepsin, or fibroblast activation protein α. A long C-terminal portion of perlecan domain IV, Dm IV-3, induced a strong clustering phenotype in the metastatic PCa cell lines, PC-3 and C4-2. MMP-7 digestion of Dm IV-3 reverses the clustering effect into one favoring cell dispersion. In a C4-2 Transwell® invasion assay, perlecan-rich human BM extract that was pre-digested with MMP-7 showed loss of barrier function and permitted a greater level of cell penetration than untreated BM extract. We conclude that enzymatic processing of perlecan in the BM or territorial matrix by MMP-7 as occurs in the invasive tumor microenvironment acts as a molecular switch to alter PCa cell behavior and favor cell dispersion and invasiveness.