High affinity binding between laminin and laminin binding protein of Leishmania is stimulated by zinc and may involve laminin zinc-finger like sequences.

In the course of trying to understand the pathogenesis of leishmaniasis in relation to extracellular matrix (ECM) elements, laminin, a major ECM protein, has been found to bind saturably and with high affinity to a 67-kDa cell surface protein of Leishmania donovani. This interaction involves a single class of binding sites, which are ionic in nature, conformation-dependent and possibly involves sulfhydryls. Binding activity was significantly enhanced by Zn2+, an effect possibly mediated through Cys-rich zinc finger-like sequences on laminin. Inhibition studies with monoclonals against polypeptide chains and specific peptides with adhesive properties revealed that the binding site was localized in one of the nested zinc finger consensus sequences of B1 chain containing the specific pentapeptide sequence, YIGSR. Furthermore, incubation of L. donovani promastigotes with C(YIGSR)3-NH2 peptide amide or antibody directed against the 67-kDa laminin-binding protein (LBP) induced tyrosine phosphorylation of proteins with a molecular mass ranging from 115 to 130 kDa. These studies suggest a role for LBP in the interaction of parasites with ECM elements, which may mediate one or more downstream signalling events necessary for establishment of infection.     

Role of 67 kDa cell surface laminin binding protein of Leishmania donovani in pathogenesis.

The role that interaction with laminin may play in Leishmania donovani infection was investigated. Binding of (125)I-radiolabeled laminin, in a liquid-phase assay, by the parasite was rapid, saturable, specific, reversible, and of high affinity. Using a Western blotting procedure, a 67 kDa laminin-binding protein (LBP) was identified from the membrane of both the promastigote and amastigote forms of L. donovani. Subsequently, the protein was purified by affinity chromatography. Immunofluorescence with a polyclonal antibody against LBP as well as flow cytometric analysis demonstrated its presence at the parasite surface. After stimulation with phorbol-12-myristate-13-acetate (PMA), U937 cells exhibited the ability to adhere to laminin and LBP specifically inhibited this adhesion. The reduced parasite adhesion after tunicamycin treatment suggested the importance of sugar residues in cell adhesion. Although co-administration of either laminin or LBP or anti LBP antibody reduced parasite virulence, resulting in a lower level of infection in the BALB/c mouse model, an in vitro macrophage culture-enhanced level of infection was observed in the case of laminin-coated parasites. The results collectively suggest a role for LBP in the interaction of the parasite with extracellular matrix elements, which may constitute a basis for the homing of the parasite to its physiological address.     

Membrane protein binding to the origin region of Bacillus subtilis.

Binding of membrane proteins extracted from Bacillus subtilis to an 11.6-kilobase region containing the origin of replication was examined by Western blotting (protein blotting) procedures. Two adjacent origin probes in the double-stranded form (spanning a length of 4 kilobases) were found to bind very strongly to a 63-kilodalton (kDa) protein in that they resisted dissociation after a high-concentration salt wash. This region encompasses both a site implicated in initiation in vivo and a gene coding for a DNA gyrase subunit (gyrA). In contrast, flanking origin and nonorigin double-stranded probes were dissociated after washing with a high salt concentration. Another protein of 67 kDa bound less intensely to the putative initiation site but not to the gyrA region. All of the origin and nonorigin probes in the double- or single-stranded form were found to bind nonspecifically to a subset of 10 to 12 proteins of 50 to 60 separated by gel electrophoresis after a low-concentration salt wash. They ranged in size from 14 to over 100 kDa (including 63 kDa). However, in contrast to the double-stranded forms, most of the single-stranded probes resisted dissociation from the protein subset after a high-concentration salt wash.     

Membrane assembly of the triple-spanning coronavirus M protein. Individual transmembrane domains show preferred orientation.

The M protein of mouse hepatitis virus strain A59 is a triple-spanning membrane protein which assembles with an uncleaved internal signal sequence, adopting an NexoCcyt orientation. To study the insertion mechanism of this protein, domains potentially involved in topogenesis were deleted and the effects analyzed in topogenesis were deleted and the effects analyzed in several ways. Mutant proteins were synthesized in a cell-free translation system in the presence of microsomal membranes, and their integration and topology were determined by alkaline extraction and by protease-protection experiments. By expression in COS-1 and Madin-Darby canine kidney-II cells, the topology of the mutant proteins was also analyzed in vivo. Glycosylation was used as a biochemical marker to assess the disposition of the NH2 terminus. An indirect immunofluorescence assay on semi-intact Madin-Darby canine kidney-II cells using domain-specific antibodies served to identify the cytoplasmically exposed domains. The results show that each membrane-spanning domain acts independently as an insertion and anchor signal and adopts an intrinsic preferred orientation in the lipid bilayer which corresponds to the disposition of the transmembrane domain in the wild-type assembled protein. These observations provide further insight into the mechanism of membrane integration of multispanning proteins. A model for the insertion of the coronavirus M protein is proposed.     

Membrane orientation and oligomerization of the melanocortin receptor accessory protein 2

The melanocortin receptor accessory protein 2 (MRAP2) plays a pivotal role in the regulation of several G protein–coupled receptors that are essential for energy balance and food intake. MRAP2 loss-of-function results in obesity in mammals. MRAP2 and its homolog MRAP1 have an unusual membrane topology and are the only known eukaryotic proteins that thread into the membrane in both orientations. In this study, we demonstrate that the conserved polybasic motif that dictates the membrane topology and dimerization of MRAP1 does not control the membrane orientation and dimerization of MRAP2. We also show that MRAP2 dimerizes through its transmembrane domain and can form higher-order oligomers that arrange MRAP2 monomers in a parallel orientation. Investigating the molecular details of MRAP2 structure is essential for understanding the mechanism by which it regulates G protein–coupled receptors and will aid in elucidating the pathways involved in metabolic dysfunction.     

Chicken gizzard 5'-nucleotidase functions as a binding protein for the laminin/nidogen complex.

Soluble and reconstituted 5'-nucleotidase were used in the binding assays to the laminin/nidogen complex. They both are shown to interact specifically and in a saturable manner with the laminin/nidogen complex using a solid-phase binding assay. Dissociation constants in the region of 10(-8) M were determined for the association of soluble and membrane-bound 5'-nucleotidase. Scatchard analysis of the binding data indicate a stoichiometry of about 2.7 of the homodimeric soluble 5'-nucleotidase to the laminin/nidogen complex. The association of 5'-nucleotidase with laminin/nidogen occurs in the absence of divalent metal ions and does not require N-linked carbohydrate moieties of both laminin/nidogen and 5'-nucleotidase. 5'-Nucleotidase also associates with isolated laminin although with reduced affinity. No binding to isolated nidogen was observed. Peptides containing the RGD sequence did not influence the binding reaction. Monoclonal and polyclonal antibodies directed against 5'-nucleotidase and laminin specifically perturb the association of the reconstituted enzyme to laminin/nidogen. Sulfated polysaccharides such as heparinsulfate and dermatansulfate modulate the interaction of 5'-nucleotidase and laminin/nidogen in a complex biphasic manner and might also regulate the binding reaction in vivo. Immunohistochemistry shows a close spatial correlation of 5'-nucleotidase and laminin also in the epithelium of the small intestine pointing to an in vivo interaction of both glycoproteins.     

Characteristics of the binding of human C-reactive protein (CRP) to laminin

Human CRP binds to the basement membrane protein laminin in vitro in a Ca2+-dependent manner via the phosphorylcholine (PC) binding site of C-reactive protein (CRP). The binding was saturable at a molar ratio of 4 (CRP/laminin). The specificity of the binding was shown by inhibition of binding of labeled CRP to laminin by unlabeled CRP, but not by human IgG. Specific binding was optimal in the presence of 5 mM Ca2+, but did not occur in the absence of Ca2+ or in the presence of EDTA. The binding of Ca2+ to CRP causes a conformational change in the molecule, which is required for binding to PC and to laminin. The PC binding site of CRP was implicated in the binding to laminin on the basis of inhibition by both soluble PC and anti-idiotypic mAbs directed to the TEPC-15 PC-binding idiotype found on mouse antibodies to PC. In addition, mouse mAbs specific for the CRP PC binding site displayed decreased reactivity with CRP already bound to laminin. The binding of CRP to laminin provides a possible explanation for selective deposition of CRP at inflamed sites. The CRP-laminin interaction may serve as a means of concentrating CRP at sites of tissue damage so that the CRP might function as a ligand for leukocytes, an event that will result in removal of necrotic tissue and cell debris.     

Membrane localization of Arabidopsis acyl-CoA binding protein ACBP2

Cytosolic acyl-CoA binding proteins bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and pool formers. Recently, we have characterized Arabidopsis thaliana cDNAs encoding novel forms of ACBP, designated ACBP1 and ACBP2, that contain a hydrophobic domain at the N-terminus and show conservation at the acyl-CoA binding domain to cytosolic ACBPs. We have previously demonstrated that ACBP1 is membrane-associated in Arabidopsis. Here, western blot analysis of anti-ACBP2 antibodies on A. thaliana protein showed that ACBP2 is located in the microsome-containing membrane fraction and in the subcellular fraction containing large particles (mitochondria, chloroplasts and peroxisomes), resembling the subcellular localization of ACBP1. To further investigate the subcellular localization of ACBP2, we fused ACBP2 translationally in-frame to GFP. By means of particle gene bombardment, ACBP2-GFP and ACBP1-GFP fusion proteins were observed transiently expressed at the plasma membrane and at the endoplasmic reticulum in onion epidermal cells. GFP fusions with deletion derivatives of ACBP1 or ACBP2 lacking the transmembrane domain were impaired in membrane targeting. Our investigations also showed that when the transmembrane domain of ACBP1 or that of ACBP2 was fused with GFP, the fusion protein was targeted to the plasma membrane, thereby establishing their role in membrane targeting. The localization of ACBP1-GFP is consistent with our previous observations using immunoelectron microscopy whereby ACBP1 was localized to the plasma membrane and vesicles. We conclude that ACBP2, like ACBP1, is a membrane protein that likely functions in membrane-associated acyl-CoA transfer/metabolism.     

Structure-function studies of the functional and binding epitope of the human 37 kDa laminin receptor precursor protein.

Expression of the 37 kDa laminin receptor precursor protein (37LRP) correlates directly with increased invasiveness and the metastatic potential of tumors. The 37LRP matures to a 67 kDa protein which facilitates the binding of cancer cells to basement membranes. The palindrome peptide sequence LMWWML, corresponding to the 173-178-residue stretch of the human 37LRP sequence, has been identified as the laminin-1-binding site. Peptides from 37LRP of species that contain this palindrome-bind laminin-1 with high affinity. Nuclear magnetic resonance (NMR) conformational studies have been undertaken on a synthetic 15-residue peptide (KGAHSVGLMWWMLAR) containing the palindrome to establish the structural basis of this activity. To further correlate the structural data with laminin-1-binding function, analogous structural studies were conducted for a similar peptide (RGKHSIGLIWYLLAR) lacking the palindrome, originating from 37LRP sequence of Saccharomyces cerevisiae and exhibiting low laminin-1-binding affinity. Finally, in vitro cell invasion assays were performed to investigate the possibility that the laminin-1-binding affinity of the peptides influences their inhibitory activity.     

Structure-guided identification of a laminin binding site on the laminin receptor precursor

The 37/ 67-kDa human laminin receptor (LamR) is a cell surface receptor for laminin, prion protein, and a variety of viruses. Because of its wide range of ligands, LamR plays a role in numerous pathologies. LamR overexpression correlates with a highly invasive cell phenotype and increased metastatic ability, mediated by interactions between LamR and laminin. In addition, the specific targeting of LamR with small interfering RNAs, blocking antibodies, and Sindbis viral vectors confers anti-tumor effects. We adopted a structure-based approach to map a laminin binding site on human LamR by comparing the sequences and crystal structures of LamR and Archaeoglobus fulgidus S2p, a non-laminin-binding ortholog. Here, we identify a laminin binding site on LamR, comprising residues Phe32, Glu35, and Arg155, which are conserved among mammalian species. Mutation of these residues results in a significant loss of laminin binding. Further, recombinant wild-type LamR is able to act as a soluble decoy to inhibit cellular migration towards laminin. Mutation of this laminin binding site results in loss of migration inhibition, which demonstrates the physiological role of Phe32, Glu35, and Arg155 for laminin binding activity. Mapping of the LamR binding site should contribute to the development of therapeutics that inhibit LamR interactions with laminin and may aid in the prevention of tumor growth and metastasis.     

Membrane orientation of sheep spectrin

Based primarily on studies of human erythrocytes, current theories of the structure and organization of erythrocyte membrane localize spectrin to the membrane cytoplasmic surface. Affinity purified anti-sheep spectrin antibodies were used in indirect immunofluorescence studies of intact erythrocytes from various vertebrate species and inside-out and right-side-out impermeable sheep erythrocyte vesicles. This investigation detected immunologically reactive external and potentially transmembranal determinant(s) of the sheep erythrocyte spectrin "assembly." Parallel studies using anti-sheep and anti-human spectrin antibodies, as well as 125I surface-labelling studies of intact sheep and human erythrocytes, indicated that this particular membrane orientation of spectrin was evident in sheep but not in human erythrocytes. Antisera containing antibodies to the external portion of this spectrin "assembly" demonstrated external fluorescence to a variable degree on some, but not all, vertebrate erythrocytes surveyed, confirming that the sheep erythrocyte was not the only exception. It is suggested that there may be subtle species variability in the intermolecular associations of the spectrin "assembly" with(in) the erythrocyte membrane not requiring alterations of the spectrin molecule itself.     

Identification and immunolocalization of the laminin binding protein from embryonic avian corneal epithelial cells

We report the identification of a 65-kDa laminin-binding protein (LBP) on the basal cell surface of embryonic corneal epithelium in chicken. The 65-kDa LBP was isolated by affinity chromatography with laminin-Sepharose. When reconstituted with lipid vesicles, it demonstrated specific binding for laminin. We produced monoclonal antibodies (MAbs) against 65-k Da LBP; these MAbs immunohistochemically localized to the basal epithelial cell surface. One MAb interfered with the binding of laminin to isolated epithelia and purified 65-k Da LBP. It appears that we have identified, at least in part, a cell-surface binding site for laminin. This site would provide the important link between the extracellular laminin and the intracellular cytoskeleton, and potentially the metabolic machinery of the corneal epithelial cell.     

Membrane binding of MARCKS-related protein studied by tryptophan fluorescence spectroscopy

MARCKS-related protein (MRP) is a peripheral membrane protein whose binding to membranes is mediated by the N-terminal myristoyl moiety and a central, highly basic effector domain. MRP mediates cross-talk between protein kinase C and calmodulin and is thought to link the actin cytoskeleton to the plasma membrane. Since MRP contains no tryptophan residues, we mutated a phenylalanine in the effector domain to tryptophan (MRP F93W) and used fluorescence spectroscopy to monitor binding of the protein to phospholipid vesicles. We report in detail the evaluation procedure necessary to extract quantitative information from the raw data. The spectra of MRP F93W obtained in the presence of increasing amounts of lipid crossed at an isosbestic point, indicating a simple transition between two states: free and membrane-bound protein. The change in fluorescence toward values typical of a more hydrophobic environment was used to quantify membrane binding. The partition coefficient agreed well with values obtained previously by other methods. To study the interaction of the N-terminus of MRP with membranes, a tryptophan residue was also introduced at position 4 (MRP S4W). Our data suggest that only the myristoylated N-terminus interacted with liposomes. These results demonstrate the versatility of site-directed incorporation of tryptophan residues to study protein-membrane interactions.     

Calcium-dependence of laminin binding to phospholipid membranes

Laminin is the most abundant noncollagenous protein in basement membranes. Its selfassembly has been studied in solution, and it has been established that calcium ions induce partially reversible aggregation. The behavior at a lipid membrane surface is of greater biological significance, but it is difficult to study quantitatively binding kinetics at a surface. The present work uses a powerful new integrated optics technique to measure the absorption and aggregation of the laminin–nidogen at a bilayer lipid membrane surface. It is found that the binding of a single layer of laminin at the lipid membrane is independent of the presence of calcium, but that the building up of multilayer laminin membranes requires calcium, and that these may not be destroyed by a calcium-complexing agent. © 1993 John Wiley & Sons, Inc.     

Mouse heart laminin. Purification of the native protein and structural comparison with Engelbreth-Holm-Swarm tumor laminin

Laminin was selectively extracted from different mouse tissues using EDTA-containing buffer. By immunoblotting with an antiserum raised against mouse Engelbreth-Holm-Swarm (EHS) tumor laminin, such extracts could be shown to contain laminin-like molecules with a low apparent proportion of A chain to B chains. Native laminin was purified from mouse heart tissue and was shown to have an aberrant polypeptide composition as compared to mouse EHS tumor laminin. Most prominently, mouse heart laminin contains an Mr 300,000 polypeptide which is not antigenically related to the A or the B chains. Furthermore, nonreducible polypeptide components were seen with apparent Mr values of 600,000 and 900,000. The Mr 600,000 component contains epitopes shared with both EHS tumor laminin and the Mr 300,000 polypeptide and possibly represents a covalently cross-linked complex of an A or B chain with the Mr 300,000 chain.     

Membrane binding kinetics of protein kinase C betaII mediated by the C2 domain.

Conventional isoforms of protein kinase C (PKC) are activated when their two membrane-targeting modules, the C1 and C2 domains, bind the second messengers diacylglycerol (DG) and Ca2+, respectively. This study investigates the mechanism of Ca2+-induced binding of PKC betaII to anionic membranes mediated by the C2 domain. Stopped-flow fluorescence spectroscopy reveals that Ca2+-induced binding of the isolated C2 domain to anionic vesicles proceeds via at least two steps: (1) rapid binding of two or more Ca2+ ions to the free domain with relatively low affinity and (2) diffusion-controlled association of the Ca2+-occupied domain with vesicles. Ca2+ increases the affinity of the C2 domain for anionic membranes by both decreasing the dissociation rate constant (k(off)) and increasing the association rate constant (k(on)) for membrane binding. For binding to vesicles containing 40 mol % anionic lipid in the presence of 200 microM Ca2+, k(off) and k(on) are 8.9 s(-1) and 1.2 x 10(10) M(-1) x s(-1), respectively. The k(off) value increases to 150 s(-1) when free Ca2+ levels are rapidly reduced, decreasing the average lifetime of the membrane-bound C2 domain (tau = k(off)(-1)) from 110 ms in the presence of Ca2+ to 6.7 ms when Ca2+ is rapidly removed. Experiments addressing the role of electrostatic interactions reveal that they stabilize either the initial C2 domain-membrane encounter complex or the high-affinity membrane-bound complex. Specifically, lowering the phosphatidylserine mole fraction or including MgCl2 in the binding reaction decreases the affinity of the C2 domain for anionic vesicles by both reducing k(on) and increasing k(off) measured in the presence of 200 microM Ca2+. These species do not affect the k(off) value when Ca2+ is rapidly removed. Studies with PKC betaII reveal that Ca2+-induced binding to membranes by the full-length protein proceeds minimally via two kinetically resolvable steps: (1) a rapid bimolecular association of the enzyme with vesicles near the diffusion-controlled limit and, most likely, (2) subsequent conformational changes of the membrane-bound enzyme. As is the case for the C2 domain, k(off) for full-length PKC betaII increases when Ca2+ is rapidly removed, reducing tau from 11 s in the presence of Ca2+ to 48 ms in its absence. Thus, both the C2 domain and the slow conformational change prolong the lifetime of the PKC betaII-membrane ternary complex in the presence of Ca2+, with rapid membrane release triggered by removal of Ca2+. These results provide a molecular basis for cofactor regulation of PKC whereby the C2 domain searches three-dimensional space at the diffusion-controlled limit to target PKC to relatively common anionic phospholipids, whereupon a two-dimensional search is initiated by the C1 domain for the more rare, membrane-partitioned DG.     

Membrane binding of a lipidated N-Ras protein studied in lipid monolayers

The adsorption of doubly lipidated full-length N-Ras protein on 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) monolayers was studied by lateral pressure analysis, grazing incidence X-ray diffraction (GIXD), and specular reflectivity (XR). N-Ras protein adsorbs to the DPPC monolayer (lateral pressure of 20 mN/m) from the subphase thereby increasing the lateral pressure in the monolayer by 4 mN/m. The protein insertion does not alter the tilt angle and structure of the lipid molecules at the air/water interface but influences the electron density profile of the monolayer. Further, electron density differences into the subphase were observed. The Fresnel normalized reflectivity could be reconstructed in the analysis using box models yielding electron density profiles of the DPPC monolayer in the absence and in the presence of N-Ras protein. The electron density profiles of the DPPC monolayer in the presence of Ras showed clear intensity variations in the headgroup/glycerol/upper chain region, the so-called interface region where previous bilayer studies had confirmed Ras binding. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Membrane binding mechanism of yeast mitochondrial peripheral membrane protein TIM44

The protein translocations across mitochondrial membranes are carried out by specialized complexes, the Translocase of Outer Membrane (TOM) and Translocase of Inner Membrane (TIM). TIM23 translocon is responsible for translocating the mitochondrial matrix proteins across the mitochondrial inner membrane. Tim44 is an essential, peripheral membrane protein in TIM23 complex. Tim44 is tightly associated with the inner mitochondrial membrane on the matrix side. The Tim44 C-Terminal Domain (CTD) functions as an Inner Mitochondrial Membrane (IMM) anchor that recruits the Presequence protein Associated Motor (PAM) to the TIM23 channel. Using X-ray crystallographic and biochemical data, we show that the N-terminal helices A1 and A2 of Tim44 - CTD are crucial for its membrane tethering function. Based on our data, we propose a model showing how the N-terminal A1 and A2 amphipathic helices can either expose their hydrophobic face during membrane binding or conceal it in the soluble form. Therefore, the A1 and A2 helices of Tim44 may function as a membrane sensor.