Inside-out signaling promotes dynamic changes in the carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) oligomeric state to control its cell adhesion properties.

Cell-cell contacts are fundamental to multicellular organisms and are subject to exquisite levels of control. The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) can engage in both cis-homophilic (parallel) oligomerization and trans-homophilic (anti-parallel) binding. In this study, we establish that the CEACAM1 transmembrane domain has a propensity to form cis-dimers via the transmembrane-embedded (432)GXXXG(436) motif and that this basal state is overcome when activated calmodulin binds to the CEACAM1 cytoplasmic domain. Although mutation of the (432)GXXXG(436) motif reduced CEACAM1 oligomerization, it did not affect surface localization of the receptor or influence CEACAM1-dependent cellular invasion by the pathogenic Neisseria. The mutation did, however, have a striking effect on CEACAM1-dependent cellular aggregation, increasing both the kinetics of cell-cell association and the size of cellular aggregates formed. CEACAM1 association with tyrosine kinase c-Src and tyrosine phosphatases SHP-1 and SHP-2 was not affected by the (432)GXXXG(436) mutation, consistent with their association with the monomeric form of wild type CEACAM1. Collectively, our results establish that a dynamic oligomer-to-monomer shift in surface-expressed CEACAM1 facilitates trans-homophilic binding and downstream effector signaling.

Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts.

SDS/PAGE is universally used in biochemistry, cell biology, and immunology to resolve minute protein amounts readily from tissue and cell extracts. Although molecular weights of water-soluble proteins are reliably determined from their SDS/PAGE mobility, most helical membrane proteins, which comprise 20-30% of the human genome and the majority of drug targets, migrate to positions that have for decades been unpredictably slower or faster than their actual formula weight, often confounding their identification. Using de novo designed transmembrane-mimetic polypeptides that match the composition of helical membrane-spanning sequences, we quantitate anomalous SDS/PAGE fractionation of helical membrane proteins by comparing the relative mobilities of these polypeptides with typical water-soluble reference proteins on Laemmli gels. We find that both the net charge and effective molecular size of the migrating particles of transmembrane-mimetic species exceed those of the corresponding reference proteins and that gel acrylamide concentration dictates the impact of these two factors on the direction and magnitude of anomalous migration. Algorithms we derived from these data compensate for this differential effect of acrylamide concentration on the SDS/PAGE mobility of a variety of natural membrane proteins. Our results provide a unique means to predict anomalous migration of membrane proteins, thereby facilitating straightforward determination of their molecular weights via SDS/PAGE.

Congenital heart block maternal sera autoantibodies target an extracellular epitope on the α1G T-type calcium channel in human fetal hearts.

BACKGROUND: Congenital heart block (CHB) is a transplacentally acquired autoimmune disease associated with anti-Ro/SSA and anti-La/SSB maternal autoantibodies and is characterized primarily by atrioventricular (AV) block of the fetal heart. This study aims to investigate whether the T-type calcium channel subunit α1G may be a fetal target of maternal sera autoantibodies in CHB. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate differential mRNA expression of the T-type calcium channel CACNA1G (α1G gene) in the AV junction of human fetal hearts compared to the apex (18-22.6 weeks gestation). Using human fetal hearts (20-22 wks gestation), our immunoprecipitation (IP), Western blot analysis and immunofluorescence (IF) staining results, taken together, demonstrate accessibility of the α1G epitope on the surfaces of cardiomyocytes as well as reactivity of maternal serum from CHB affected pregnancies to the α1G protein. By ELISA we demonstrated maternal sera reactivity to α1G was significantly higher in CHB maternal sera compared to controls, and reactivity was epitope mapped to a peptide designated as p305 (corresponding to aa305-319 of the extracellular loop linking transmembrane segments S5-S6 in α1G repeat I). Maternal sera from CHB affected pregnancies also reacted more weakly to the homologous region (7/15 amino acids conserved) of the α1H channel. Electrophysiology experiments with single-cell patch-clamp also demonstrated effects of CHB maternal sera on T-type current in mouse sinoatrial node (SAN) cells. CONCLUSIONS/SIGNIFICANCE: Taken together, these results indicate that CHB maternal sera antibodies readily target an extracellular epitope of α1G T-type calcium channels in human fetal cardiomyocytes. CHB maternal sera also show reactivity for α1H suggesting that autoantibodies can target multiple fetal targets.

Design of transmembrane peptides: coping with sticky situations.

Membrane proteins have central roles in cellular processes ranging from nutrient uptake to cell-cell communication, and are key drug targets. However, research on α-helical integral membrane proteins is in its relative infancy vs. water-soluble proteins, largely because of their water insolubility when extracted from their native membrane environment. Peptides with sequences that correspond to the membrane-spanning segments of α-helical integral membrane proteins, termed transmembrane (TM) peptides, provide valuable tools for the characterization of these molecules. Here we describe in detail protocols for the design of TM peptides from the sequences of natural α-helical integral membrane proteins and outline strategies for their synthesis and for improving their solubility properties.

Sequence hydropathy dominates membrane protein response to detergent solubilization.

The ability to predict from amino acid sequence how membrane protein structures will respond to detergent solubilization would significantly facilitate experimental characterization of these molecules. Here we have investigated and compared the response to solubilization by the "mild" n-dodecyl-ß-D-maltoside (DDM) and "harsh" sodium dodecyl sulfate (SDS) of wild-type and point mutant "hairpin" (helix-loop-helix) membrane proteins derived from the third and fourth TM segments of the human cystic fibrosis transmembrane conductance regulator (CFTR) and the intervening extracellular loop. Circular dichroism spectroscopy, size-exclusion chromatography, and pyrene fluorescence spectroscopy were used to evaluate the secondary structures, hairpin-detergent complex excluded volumes, and hairpin compactness of the detergent-solubilized sequences. Sequence hydrophobicity is found to be the dominant factor dictating membrane protein response to detergent solubilization by DDM and SDS, with hairpin secondary structure exquisitely sensitive to mutation when DDM is used for solubilization. DDM and SDS differ principally in their ability to promote approach of TM segment ends, although hairpin compactness remains sensitive to point mutations. Our overall findings suggest that protein-protein and protein-detergent interactions are determined concomitantly, with the net hydropathy of residues exposed to detergent dominating the observed properties of the solubilized protein.

Protein structure in membrane domains.

Of great interest to the academic and pharmaceutical research communities, helical transmembrane proteins are characterized by their ability to dissolve and fold in lipid bilayers--properties conferred by polypeptide spans termed transmembrane domains (TMDs). The apolar nature of TMDs necessitates the use of membrane-mimetic solvents for many structure and folding studies. This review examines the relationship between TMD structure and solvent environment, focusing on principles elucidated largely in membrane-mimetic environments with single-TMD protein and peptide models. Following a brief description of TMD sequence and conformational characteristics gleaned from the structural database, we present an overview of the conceptual models used to study folding in vitro. The impact of sequence and solvent context on the incorporation of TMDs into membranes, and its role in measurements of TMD self-assembly strengths, is then described. We conclude with a discussion of the nonspecific effects of membrane components on TMD stability.

Structural, functional, and bioinformatic studies demonstrate the crucial role of an extended peptide binding site for the SH3 domain of yeast Abp1p.

SH3 domains, which are among the most frequently occurring protein interaction modules in nature, bind to peptide targets ranging in length from 7 to more than 25 residues. Although the bulk of studies on the peptide binding properties of SH3 domains have focused on interactions with relatively short peptides (less than 10 residues), a number of domains have been recently shown to require much longer sequences for optimal binding affinity. To gain greater insight into the binding mechanism and biological importance of interactions between an SH3 domain and extended peptide sequences, we have investigated interactions of the yeast Abp1p SH3 domain (AbpSH3) with several physiologically relevant 17-residue target peptide sequences. To obtain a molecular model for AbpSH3 interactions, we solved the structure of the AbpSH3 bound to a target peptide from the yeast actin patch kinase, Ark1p. Peptide target complexes from binding partners Scp1p and Sjl2p were also characterized, revealing that the AbpSH3 uses a common extended interface for interaction with these peptides, despite K(d) values for these peptides ranging from 0.3 to 6 mum. Mutagenesis studies demonstrated that residues across the whole 17-residue binding site are important both for maximal in vitro binding affinity and for in vivo function. Sequence conservation analysis revealed that both the AbpSH3 and its extended target sequences are highly conserved across diverse fungal species as well as higher eukaryotes. Our data imply that the AbpSH3 must bind extended target sites to function efficiently inside the cell.

Peptide models of membrane protein folding.

Given the central roles of membrane proteins in cellular processes ranging from nutrient uptake to cell-cell communication, as well as the importance of these proteins as drug targets, efforts to understand and control their structures are vital in human health and disease. The rational design of membrane proteins with modified properties is thus a highly desirable goal in molecular medicine and biotechnology. However, experimental data showing how individual transmembrane (TM) residues and/or segments direct the packing and folding of membrane proteins into biologically functional entities remain sparse. To address these questions in a systematic manner, helix-helix interactions between two (or more) TM segments must be identified and analyzed. Here we present an overview of the utilization of peptides as models of the TM segments of alpha-helical membrane proteins in uncovering the amino acid sequence motifs and interactions that build these molecules. TM peptide design and production strategies are discussed, and specific examples of the application of TM peptides to the study of membrane proteins are presented. We demonstrate that TM peptides can be routinely produced in sufficient quantities for biophysical analysis, are amenable to a variety of experimental techniques, and can effectively replicate the native helix-helix contacts and key aspects of the natural biological structures of membrane proteins.

Detergent binding explains anomalous SDS-PAGE migration of membrane proteins.

Migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that does not correlate with formula molecular weights, termed "gel shifting," appears to be common for membrane proteins but has yet to be conclusively explained. In the present work, we investigate the anomalous gel mobility of helical membrane proteins using a library of wild-type and mutant helix-loop-helix ("hairpin") sequences derived from transmembrane segments 3 and 4 of the human cystic fibrosis transmembrane conductance regulator (CFTR), including disease-phenotypic residue substitutions. We find that these hairpins migrate at rates of -10% to +30% vs. their actual formula weights on SDS-PAGE and load detergent at ratios ranging from 3.4-10 g SDS/g protein. We additionally demonstrate that mutant gel shifts strongly correlate with changes in hairpin SDS loading capacity (R(2) = 0.8), and with hairpin helicity (R(2) = 0.9), indicating that gel shift behavior originates in altered detergent binding. In some cases, this differential solvation by SDS may result from replacing protein-detergent contacts with protein-protein contacts, implying that detergent binding and folding are intimately linked. The CF-phenotypic V232D mutant included in our library may thus disrupt CFTR function via altered protein-lipid interactions. The observed interdependence between hairpin migration, SDS aggregation number, and conformation additionally suggests that detergent binding may provide a rapid and economical screen for identifying membrane proteins with robust tertiary and/or quaternary structures.

Recognition of non-canonical peptides by the yeast Fus1p SH3 domain: elucidation of a common mechanism for diverse SH3 domain specificities.

The yeast Fus1p SH3 domain binds to peptides containing the consensus motif, R(S/T)(S/T)SL, which is a sharp contrast to most SH3 domains, which bind to PXXP-containing peptides. Here, we have demonstrated that this domain binds to R(S/T)(S/T)SL-containing peptides derived from two putative in vivo binding partners from yeast proteins, Bnr1p and Ste5p, with K(d) values in the low micromolar range. The R(S/T)(S/T)SL consensus motif is necessary, but not sufficient for binding to the Fus1p SH3 domain, as residues lying N-terminal to the consensus motif also play a critical role in the binding reaction. Through mutagenesis studies and comparisons to other SH3 domains, we have discovered that the Fus1p SH3 domain utilizes a portion of the same binding surface as typical SH3 domains. However, the PXXP-binding surface, which plays the predominant role in binding for most SH3 domains, is debilitated in the WT domain by the substitution of unusual residues at three key conserved positions. By replacing these residues, we created a version of the Fus1p SH3 domain that binds to a PXXP-containing peptide with extremely high affinity (K(d)= 40 nM). Based on our data and analysis, we have clearly delineated two distinct surfaces comprising the typical SH3-domain-binding interface and show that one of these surfaces is the primary mediator of almost every "non-canonical" SH3-domain-mediated interaction described in the literature. Within this framework, dramatic alterations in SH3 domain specificity can be simply explained as a modulation of the binding strengths of these two surfaces.

Defining the defect in F508 del CFTR: a soluble problem?

Previously reported crystal structures of CFTR F508 del-NBD1 were determined in the presence of solubilizing mutations. In this issue of Chemistry & Biology, Pissarra et al. (2008) show that partial rescue of the trafficking and gating defects of full-length CFTR occurs in vivo upon recapitulation of the solubilizing F494N/Q637R or F428S/F494N/Q637R substitutions in cis with F508 del.

Surface recognition elements of membrane protein oligomerization.

Although certain membrane proteins are functional as monomeric polypeptides, others must assemble into oligomers to carry out their biological roles. High-resolution membrane protein structures provide a valuable resource for examining the sequence features that facilitate-or preclude-assembly of membrane protein monomers into multimeric structures. Here we have utilized a data set of 28 high-resolution alpha-helical membrane protein structures comprising 32 nonredundant polypeptides to address this issue. The lipid-exposed surfaces of membrane proteins that have reached their fully assembled and functional biological units have been compared with those of the individual subunits that build quaternary structures. Though the overall amino acid composition of each set of surfaces is similar, a key distinction-the distribution of small-xxx-small motifs-delineates subunits from membrane proteins that have reached a functioning oligomeric state. Quaternary structure formation may therefore be dictated by small-xxx-small motifs that are not satisfied by intrachain contacts.

Role of the extracellular loop in the folding of a CFTR transmembrane helical hairpin.

The folding of membrane-spanning domains into their native functional forms depends on interactions between transmembrane (TM) helices joined by covalent loops. However, the importance of these covalent linker regions in mediating the strength of helix-helix associations has not been systematically addressed. Here we examine the potential structural impact of cystic fibrosis-phenotypic mutations in the extracellular loop 2 (ECL2) on interactions between the TM3 and TM4 helices of the cystic fibrosis transmembrane conductance regulator (CFTR) in constructs containing CFTR residues 194-241. When the effects of replacements in ECL2 (including the CF-phenotypic mutants E217G and Q220R) were evaluated in a library of wild-type and mutant TM3-ECL2-TM4 hairpin constructs, we found that SDS-PAGE gel migration rates differed over a range of nearly 40% +/- the wild-type position and that decreased migration rates correlate with increasing hairpin alpha-helical content as measured by circular dichroism spectra in sodium dodecyl sulfate micelles. The decreased mobility of TM3/4 constructs by introduction of non-native residues is interpreted in terms of an elongation or "opening" of the helical hairpin and concomitant destabilization of membrane-based helix-helix interactions. Our results support a role for short loop regions in dictating the stability of membrane protein folds and highlight the interplay between membrane-embedded helix-helix interactions and loop conformation in influencing the structure of membrane proteins.

Membrane protein assembly patterns reflect selection for non-proliferative structures.

Membrane proteins that regulate solute movement are often built from multiple copies of an identical polypeptide chain. These complexes represent striking examples of self-assembling systems that recruit monomers only until a prescribed level for function is reached. Here we report that three modes of assembly - distinguished by sequence and stoichiometry - describe all helical membrane protein complexes currently solved to high resolution. Using the 13 presently available non-redundant homo-oligomeric structures, we show that two of these types segregate with protein function: one produces energy-dependent transporters, while the other builds channels for passive diffusion. Given such limited routes to functional complexes, membrane proteins that self-assemble exist on the edge of aggregation, susceptible to mutations that may underlie human diseases.

Peptides as transmembrane segments: decrypting the determinants for helix-helix interactions in membrane proteins.

Although the structural analysis of membrane proteins is advancing, an understanding of the basic principles that underlie their folding and assembly remains limited because of the high insolubility intrinsic to these molecules and concomitant challenges in obtaining crystals. Fortunately, from an experimental standpoint, membrane protein folding can be approximated as the rigid-body docking of pre-formed alpha-helical transmembrane segments one with another to form the final functional protein structure. Peptides derived from the sequences of native alpha-helical transmembrane segments and those that mimic their properties are therefore valuable in the experimental evaluation of protein folding within the membrane. Here we present an overview of the progress made in our laboratory and elsewhere in using peptide models toward defining the sequence requirements and forces stabilizing membrane protein folds.

Lipid solvation effects contribute to the affinity of Gly-xxx-Gly motif-mediated helix-helix interactions.

Interactions between transmembrane helices are mediated by the concave Gly-xxx-Gly motif surface. Whether Gly residues per se are sufficient for selection of this motif has not been established. Here, we used the in vivo TOXCAT assay to measure the relative affinities of all 18 combinations of Gly, Ala, and Ser "small-xxx-small" mutations in glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP) homodimers. Affinity values were compared with the accessibility to a methylene-sized probe of the total surface area of each helix monomer as a measure of solvation by membrane components. A strong inverse correlation was found between nonpolar-group lipid accessibility and dimer affinity (R = 0.75 for GpA, p = 0.013, and R = 0.81 for MCP, p = 0.004), suggesting that lipid as a poor membrane protein solvent, conceptually analogous to water in soluble protein folding, can contribute to dimer stability and help to define helix-helix interfaces.

The structure of "unstructured" regions in peptides and proteins: role of the polyproline II helix in protein folding and recognition.

Classical descriptions of the three-dimensional shapes of proteins usually invoke three main structures: alpha-helix, beta-sheet, and beta-turn. More recently, the polyproline II (PPII) structure has been implicated in diverse biological activities including signal transduction, transcription, cell motility, and immune response. Concurrently, evidence is accumulating that PPII structure has a significant role in the unfolded states of proteins. In this article, we connect the structural properties of PPII helices to their roles in protein recognition and protein unfolded states. The properties unique to the PPII conformation are linked to the exploitation of this structure for the molecular recognition of proteins, using peptide ligands of the Src homology 3 (SH3) domain as an example. The evidence supporting a role for PPII conformation in protein-unfolded states is also presented in the context of the forces that may stabilize the PPII conformation in unfolded polypeptides.