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M A Lemmon

Publications and source records attributed to M A Lemmon.

49 records · Page 3Linked to original sources
PubMed1994

Crystal structure at 2.2 A resolution of the pleckstrin homology domain from human dynamin.

The X-ray crystal structure of the pleckstrin homology (PH) domain from human dynamin has been refined to 2.2 A resolution. A seven-stranded beta sandwich of two orthogonal antiparallel beta sheets is closed at one corner by a C-terminal alpha helix. Opposite this helix are the three loops that vary most among PH domains. The basic fold is very similar to that of two other PH domains recently determined by nuclear magnetic resonance, confirming that PH domain with known structure is electrostatically polarized, with the three variable loops forming a positively charged surface. This surface includes the position of the X-linked immunodeficiency mutation in the Btk PH domain and may serve as a ligand-binding surface.

Amino Acid Sequence↗
PubMed1994

Structural organization of the pentameric transmembrane alpha-helices of phospholamban, a cardiac ion channel.

Phospholamban is a 52 amino acid calcium regulatory protein found as pentamers in cardiac SR membranes. The pentamers form through interactions between its transmembrane domains, and are stable in SDS. We have employed a saturation mutagenesis approach to study the detailed interactions between the transmembrane segments, using a chimeric protein construct in which staphylococcal nuclease (a monomeric soluble protein) is fused to the N-terminus of phospholamban. The chimera forms pentamers observable in SDS-PAGE, allowing the effects of mutations upon the oligomeric association to be determined by electrophoresis. The disruptive effects of amino acid substitutions in the transmembrane domain were classified as sensitive, moderately sensitive or insensitive. Residues of the same class lined up on faces of a 3.5 amino acids/turn helical projection, allowing the construction of a model of the interacting surfaces in which the helices are associated in a left-handed pentameric coiled-coil configuration. Molecular modeling simulations (to be described elsewhere in detail) confirm that the helices readily form a left-handed coiled-coil helical bundle and have yielded molecular models for the interacting surfaces, the best of which is identical to that predicted by the mutagenesis. Residues lining the pore show considerable structural sensitivity to mutation, indicating that care must be taken in interpreting the results of mutagenesis studies of channels. The cylindrical ion pore (minimal diameter of 2 A) appears to be defined largely by hydrophobic residues (I40, L43 and I47) with only two mildly polar elements contributed by sulfurs in residues C36 and M50.

Amino Acid Sequence↗
PubMed1994

Specificity and promiscuity in membrane helix interactions.

Transmembrane alpha-helices can associate with one another in lipid bilayers. This association is important in the folding and oligomerization of many integral membrane proteins, and may also play a role in their function. The interactions between helices may be highly specific or relatively non-specific, and their roles may differ accordingly. These two cases are discussed.

Amino Acid Sequence↗
PubMed1994

Thermodynamic studies of tyrosyl-phosphopeptide binding to the SH2 domain of p56lck.

The interaction between SH2 domains and tyrosine-phosphorylated proteins is essential in several cytosolic signal transduction pathways. Here we report thermodynamic studies of the interaction of the p56lck (lck) SH2 domain with several phosphopeptides, using the technique of isothermal titration calorimetry (ITC). This is the first report of the use of ITC to study SH2 domain binding reactions. The free energy of binding of the SH2 domain of lck to a phosphopeptide corresponding to the autoregulatory C-terminus of the protein (pY505) was found to be similar to that measured for a phosphopeptide modeled on the C-terminus of the epidermal growth-factor receptor (delta G degrees approximately -7.0 kcal mol-1 at pH 6.8), although significant differences in the enthalpy and entropy were observed. Binding of a phosphopeptide modeled on the C-terminus of p185neu was weaker (delta G degrees approximately -5.4 kcal mol-1 at pH 6.8). Lowering the pH to 5.5 reduced the binding affinity of pY505 by approximately 1 order of magnitude. We ascribe this to the protonation of a histidine side chain in the SH2 domain (H180), which is involved in a hydrogen-bonding network that optimizes the binding site geometry. No difference in affinity was observed between portions of lck corresponding to the SH3-SH2 (residues 63-228) and SH2 (residues 123-228) domains for the pY505 peptide. We also studied the effect upon pY505 peptide binding of mutations at two highly conserved arginine residues in the lck SH2 domain (R134 and R154).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Stabilization of an active dimeric form of the epidermal growth factor receptor by introduction of an inter-receptor disulfide bond.

Populations of the epidermal growth factor receptor (EGFR) with both high and low affinity for EGF are found on the surface of cells. It has been hypothesized that the high-affinity state of the EGFR represents the receptor dimer and that this is also the kinase-active form. We describe here studies aimed at addressing this question directly. To stabilize homodimers of EGFR, we have generated a mutated form of the receptor by inserting a cysteine residue in the extracellular juxtamembranous region, in order to cross-link the extracellular domains of two receptors via disulfide bond formation. The mutation resulted in ligand-induced appearance of covalently linked EGF receptor dimers and, in parallel, increased the number of high-affinity receptors present on the surface of cells expressing the mutated EGFR. Comparison of the tyrosine kinase activity of the covalently linked dimeric and the monomeric forms of the EGF receptor, separated by sucrose density gradient centrifugation, showed that the dimer was significantly more active than monomer in the phosphorylation of exogenous substrate. We conclude that the dimeric form of the EGF-receptor represents the active form, and that dimer formation is associated with the appearance of high-affinity binding EGF receptors on the cell surface.

3T3 Cells↗
PubMed1994

Regulation of signal transduction and signal diversity by receptor oligomerization.

Receptor oligomerization was initially proposed as a mechanism by which epidermal growth factor activates the protein tyrosine kinase activity of its receptor. It is now well established that ligand-induced receptor oligomerization plays an important role in transmembrane signaling by a large number of receptors for hormones, cytokines and growth factors. Heterodimerization of the extracellular domains of two members of the same receptor family, or interaction with an accessory molecule, can increase the diversity of ligands recognized by individual receptors. Heterodimerization of cytoplasmic domains permits the recruitment of different complements of SH2-domain-containing signaling molecules, increasing the repertoire of signaling pathways that can be activated by a given receptor.

Biopolymers↗
PubMed1994

A dimerization motif for transmembrane alpha-helices.

Specific helix-helix interactions inside lipid bilayers guide the folding and assembly of many integral membrane proteins and their complexes. We report here a pattern of 7 amino acids (LIxxGVxxGVxxT) which when introduced into several hydrophobic transmembrane alpha-helices promotes their specific dimerization. Dimerization is driven by interactions that are specific, dominated by the helix-helix interface, and involve no potentially ionizable groups. The motif may provide a useful tool for the functional analysis of such interactions in a variety of systems. Further, since this particular motif is rare, whilst specific helix association is not, many other such motifs may exist, which could permit sorting within complex membranes as well as guiding folding and oligomerization.

Amino Acid Sequence↗
PubMed1992

Sequence specificity in the dimerization of transmembrane alpha-helices.

While several reports have suggested a role for helix-helix interactions in membrane protein oligomerization, there are few direct biochemical data bearing on this subject. Here, using mutational analysis, we show that dimerization of the transmembrane alpha-helix of glycophorin A in a detergent environment is spontaneous and highly specific. Very subtle changes in the side-chain structure at certain sensitive positions disrupt the helix-helix association. These sensitive positions occur at approximately every 3.9 residues along the helix, consistent with their comprising the interface of a closely fit transmembranous supercoil of alpha-helices. By contrast with other reported cases of interactions between transmembrane helices, the set of interfacial residues in this case contains no highly polar groups. Amino acids with aliphatic side chains define much of the interface, indicating that precise packing interactions between the helices may provide much of the energy for association. These data highlight the potential general importance of specific interactions between the hydrophobic anchors of integral membrane proteins.

Amino Acid Sequence↗
PubMed1992

The glycophorin A transmembrane domain dimer: sequence-specific propensity for a right-handed supercoil of helices.

Recent studies suggest specific roles for transmembrane helix association in a range of functions, but understanding of the conformation and energetics of these interactions has been elusive. We have studied the specific dimerization of the transmembrane helix of glycophorin A by calculating the minimized interaction energies of a large number of conformations using simulated annealing techniques and tested the models against mutational analysis data. We find that the dimer is best modeled as a right-handed supercoil with an extensive region of close packing along the dimer interface. Furthermore, we observe a sequence-specific propensity for a right-handed supercoil to form when starting the simulated annealing modeling from a dimer of helices with parallel axes, in contrast with the dimerization region of the transcription factor GCN4 which shows a high propensity for the more prevalent left-handed supercoiling.

Amino Acid Sequence↗
PubMed1992

Glycophorin A dimerization is driven by specific interactions between transmembrane alpha-helices.

Specific side-by-side interactions between transmembrane alpha-helices may be important in the assembly and function of integral membrane proteins. We describe a system for the genetic and biophysical analysis of these interactions. The transmembrane alpha-helical domain of interest is fused to the C-terminus of staphylococcal nuclease. The resulting chimera can be expressed at high levels in Escherichia coli and is readily purified. In our initial application we study the single transmembrane alpha-helix of human glycophorin A (GpA), thought to mediate the SDS-stable dimerization of this protein. The resulting chimera forms a dimer in SDS, which is disrupted upon addition of a peptide corresponding to the transmembrane domain of GpA. Deletion mutagenesis has been used to delineate the minimum transmembrane domain sufficient for this behavior. Site-specific mutagenesis shows that a methionine residue, previously implicated as a potential interfacial residue, can be replaced with other hydrophobic residues without disrupting dimerization. By contrast, rather conservative substitutions at a valine on a different face of the alpha-helix disrupt dimerization, suggesting a high degree of specificity in the helix-helix interactions. This approach allows the interface between interacting helices to be defined.

Amino Acid Sequence↗
PubMedDate not supplied

Phosphatidylinositol-4,5-bisphosphate is required for endocytic coated vesicle formation.

Receptor-mediated endocytosis via clathrin-coated vesicles has been extensively studied and, while many of the protein players have been identified, much remains unknown about the regulation of coat assembly and the mechanisms that drive vesicle formation [1]. Some components of the endocytic machinery interact with inositol polyphosphates and inositol lipids in vitro, implying a role for phosphatidylinositols in vivo [2] [3]. Specifically, the adaptor protein complex AP2 binds phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), PtdIns(3)P, PtdIns(3,4,5)P3 and inositol phosphates. Phosphatidylinositol binding regulates AP2 self-assembly and the interactions of AP2 complexes with clathrin and with peptides containing endocytic motifs [4] [5]. The GTPase dynamin contains a pleckstrin homology (PH) domain that binds PtdIns(4,5)P2 and PtdIns(3,4,5)P3 to regulate GTPase activity in vitro [6] [7]. However, no direct evidence for the involvement of phosphatidylinositols in clathrin-mediated endocytosis exists to date. Using well-characterized PH domains as high affinity and high specificity probes in combination with a perforated cell assay that reconstitutes coated vesicle formation, we provide the first direct evidence that PtdIns(4,5)P2 is required for both early and late events in endocytic coated vesicle formation.

Adaptor Protein Complex 2↗