Biomedical subjects

Karen G Fleming

Publications and source records attributed to Karen G Fleming.

11 recordsLinked to original sources

PubMed2006

Dimerization of the erythropoietin receptor transmembrane domain in micelles.

Erythropoietin receptor (EpoR) homodimerization is an initial regulatory step in erythrocyte formation. Receptor dimers form before ligand binding, suggesting that association between receptor proteins is dependent on the receptor itself. EpoR dimerization is an essential step in erythropoiesis, and misregulation of this dimerization has been implicated in several disease states, including multi-lineage leukemias; nevertheless, how EpoR regulates its own dimerization is unclear. In vivo experiments suggest the single-pass transmembrane helix is the strongest candidate for driving ligand-independent association. To address the self-association potential of this transmembrane segment, we studied its interaction energetics in micelles by utilizing a previously successful Staphylococcal nuclease (SN-EpoR TM) fusion protein. This fusion protein strategy allows expression of the EpoR transmembrane domain in Escherichia coli independent of the other EpoR domains. Sedimentation equilibrium analytical ultracentrifugation of the detergent-solubilized SN-EpoR TM demonstrated that the murine EpoR transmembrane domain self-associates to form dimers. Although this interaction is not as stable as the dimerization of the well-studied glycophorin A transmembrane dimer, the murine EpoR transmembrane domain dimer is more stable than the interactions of the colon carcinoma kinase 4 transmembrane domain. The same experiments with the human EpoR transmembrane domain, which differs from the mouse sequence by only three residues, revealed a less favorable interaction than that of the murine sequence and is only slightly more favorable than that expected for non-preferential binding. These results suggest that the mouse and human receptor proteins may differ in the roles they play in signaling.

Amino Acid Sequence↗

PubMed2006

Lipid chain selectivity by outer membrane phospholipase A.

Outer membrane phospholipase A (OMPLA) is a unique, integral membrane enzyme found in Gram-negative bacteria and is an important virulence factor for pathogens such as Helicobacter pylori. This broad-specificity lipase degrades a variety of lipid substrates, and it plays a direct role in adjusting the composition and permeability of bacterial membranes under conditions of stress. Interestingly, OMPLA shows little preference for the lipid headgroup and, instead, the length of the hydrophobic acyl chain is the strongest determinant for substrate selection by OMPLA, with the enzyme strongly preferring substrates with chains equal to or longer than 14 carbon atoms. The question remains as to how a hydrophobic protein like OMPLA can achieve this specificity, particularly when the shorter chains can be accommodated in the binding pocket. Using a series of sulfonyl fluoride inhibitors with various lengths of acyl chain, we show here that the thermodynamics of substrate-induced OMPLA dimerization are guided by the acyl chain length, demonstrating that OMPLA uses a unique biophysical mechanism to select its phospholipid substrate.

Bacterial Outer Membrane Proteins↗

PubMed2006

Energetics of outer membrane phospholipase A (OMPLA) dimerization.

Outer membrane phospholipase A (OMPLA) is a widely conserved transmembrane enzyme found in Gram-negative bacteria, and it is implicated in the virulence of a number of pathogenic organisms. The regulation of the protein's phospholipase activity is not well understood despite the existence of a number of high resolution structures. Previous biochemical studies have demonstrated that dimerization of OMPLA is a prerequisite for its phospholipase activity, and it has been shown in vitro that this dimerization is dependent on calcium and substrate binding. Therefore, to fully understand the regulation of OMPLA, it is necessary to understand the stability of the protein dimer and the extent to which it is influenced by its effector molecules. We have used sedimentation equilibrium analytical ultracentrifugation to dissect the energetics of Escherichia coli OMPLA dimerization in detergent micelles. We find that calcium contributes relatively little stability to the dimer, while interactions with the substrate acyl chain are the predominant force in stabilizing the dimeric conformation of the enzyme. The resulting thermodynamic cycle suggests that interactions between effector molecules are additive. These energetic measurements not only provide insight into the activation of OMPLA, but they also represent the first quantitative investigation of the association energetics of a transmembrane beta-barrel. This thermodynamic study allows us to begin to address the differences between protein-protein interfaces in transmembrane proteins with a helical fold to those of a beta-barrel fold and to more fully understand the forces involved in membrane protein interactions.

Bacterial Outer Membrane Proteins↗

PubMed2006

Alternate interfaces may mediate homomeric and heteromeric assembly in the transmembrane domains of SNARE proteins.

The fusion of a vesicle to a target membrane is mediated by temporally and spatially regulated interactions within a set of evolutionarily conserved proteins. Integral to proper fusion is the interaction between proteins originating on both vesicle and target membranes to form a protein bridge between the two membranes, known as the SNARE complex. This protein complex includes the single-pass transmembrane helix proteins: syntaxin and synaptobrevin. Experimental data and amino acid sequence analysis suggest that an interface of interaction is conserved between the transmembrane regions of the two proteins. However, conflicting reports have been presented on the role of the synaptobrevin transmembrane domain in mediating important protein-protein interactions. To address this question, a thermodynamic study was carried out to determine quantitatively the self-association propensities of the transmembrane domains of synaptobrevin and syntaxin. Our results show that the transmembrane domain of synaptobrevin has only a modest ability to self-associate, whereas the transmembrane domain of syntaxin is able to form stable homodimers. Nevertheless, by a single amino acid substitution, synaptobrevin can be driven to dimerize with the same affinity as syntaxin. Furthermore, crosslinking studies show that dimerization of synaptobrevin is promoted by oxidizing agents. Despite the presence of a conserved cysteine residue in the same location as in synaptobrevin, syntaxin dimerization is not promoted by oxidization. This analysis suggests that subtle yet distinct differences are present between the two transmembrane dimer interfaces. A syntaxin/synaptobrevin heterodimer is able to form under oxidizing conditions, and we propose that the interface of interaction for the heterodimer may resemble the homodimer interface formed by the synaptobrevin transmembrane domain. Computational analysis of the transmembrane sequences of syntaxin and synaptobrevin reveal structural models that correlate with the experimental data. These data may provide insight into the role of transmembrane segments in the mechanism of vesicle fusion.

Animals↗

PubMed2005

The transmembrane domains of ErbB receptors do not dimerize strongly in micelles.

The epidermal growth factor receptors (erbB) constitute an important class of single pass transmembrane receptors involved in the transduction of signals important for cell proliferation and differentiation. Receptor association is a key event in the signal transduction process, but the molecular basis of this interaction is not fully understood. Previous biochemical and genetic studies have suggested that the single transmembrane helices of these receptor proteins might play a role in stabilizing the receptor complexes. To determine if the erbB transmembrane domains could provide a driving force to stabilize the receptor dimers, we carried out a thermodynamic study of these domains expressed as C-terminal fusion proteins with staphylococcal nuclease. Similar fusion constructs have been used successfully to investigate the oligomerization and association thermodynamics of a number of transmembrane sequences, including that of glycophorin A. Using SDS-PAGE analysis and sedimentation equilibrium analytical ultracentrifugation, we do not find strong, specific homo or hetero-interactions between the transmembrane domains of the erbB receptors in micellar solutions. Our results indicate that any preferential interactions between these domains in micellar solutions are extremely modest, of the order of 1 kcal mol(-1) or less. We applied a thermodynamic formalism to assess the effect of weakly interacting TM segments on the behavior of a covalently attached soluble domain. In the case of the ligand-bound EGFR ectodomain, we find that restriction of the ectodomain to the micellar phase by a hydrophobic TM, even in the absence of strong specific interactions, is largely sufficient to account for the previously reported increase in dimerization affinity.

Amino Acid Sequence↗

PubMed2005

The GxxxG-containing transmembrane domain of the CCK4 oncogene does not encode preferential self-interactions.

The recently cloned colon carcinoma kinase 4 (CCK4) oncogene contains an evolutionarily conserved GxxxG motif in its single transmembrane domain (TMD). It has previously been suggested that this pairwise glycine motif may provide a strong driving force for transmembrane helix-helix interactions. Since CCK4 is thought to represent a new member of the receptor tyrosine kinase family, interactions between the TMDs may be important in receptor self-association and activation of signal transduction pathways. To determine whether this conserved CCK4 TMD can drive protein-protein interactions, we have carried out a thermodynamic study using the TMD expressed as a Staphylococcal nuclease (SN) fusion protein. Similar SN-TMD fusion proteins have been used to determine the sequence specificity and thermodynamics of transmembrane helix-helix interactions in a number of membrane proteins, including glycophorin A. Using sedimentation equilibrium in C14 betaine micelles, we discovered that the CCK4 TMD is unable to drive strong protein-protein interactions. At high protein/detergent ratios, the SN-CCK4 fusion protein will dimerize, but a stochastic model for protein association in micelles can explain the observed dimer population. For low-affinity interactions such as the one studied here, an understanding of this discrete stochastic distribution of membrane proteins in micelles is important for distinguishing between preferential and random self-interactions, which can both influence the oligomeric population. The lack of a thermodynamically meaningful self-association propensity for the CCK4 TMDs demonstrates that a GxxxG motif is not sufficient to drive transmembrane helix-helix interactions.

Amino Acid Motifs↗

PubMed2004

Complex interactions at the helix-helix interface stabilize the glycophorin A transmembrane dimer.

To explore the residue interactions in the glycophorin A dimerization motif, an alanine scan double mutant analysis at the helix-helix interface was carried out. These data reveal a combination of additive and coupled effects. The majority of the double mutants are found to be equally or slightly more stable than would be predicted by the sum of the energetic cost of the single-point mutants. The proximity of the mutated sites is not related to the presence of coupling between those sites. Previous studies reveal that a single face of the glycophorin A monomer contains a specific glycine-containing motif (GxxxG) that is thought to be a driving force for the association of transmembrane helices. Double mutant cycles suggest that the relationship of the GxxxG motif to the remainder of the helix-helix interface is complex. Sequences containing mutations that abolish the GxxxG motif retain an ability to dimerize, while a sequence containing a GxxxG motif appears unable to form dimers. The energetic effects of weakly coupled and additive double mutants can be explained by changes in van der Waals interactions at the dimer interface. These results emphasize the fact that the sequence context of the dimer interface modulates the strength of the glycophorin A GxxxG-mediated transmembrane dimerization reaction.

Amino Acid Motifs↗

PubMed2004

Binding of polyubiquitin chains to ubiquitin-associated (UBA) domains of HHR23A.

Ubiquitin-associated (UBA) domains are small protein domains that occur in the context of larger proteins and are likely to function as inter- and intramolecular communication elements in ubiquitin/polyubiquitin signaling. Although monoubiquitin/UBA complexes are well characterized, much less is known about UBA/polyubiquitin complexes, even though polyubiquitin chains are believed to be biologically relevant ligands of many UBA domain proteins. Here, we report the results of a quantitative study of the interaction of K48-linked polyubiquitin chains with UBA domains of the DNA repair/proteolysis protein HHR23A, using surface plasmon resonance and other approaches. We present evidence that the UBL domain of HHR23A negatively regulates polyubiquitin/UBA interactions and identify leucine 8 of ubiquitin as an important determinant of chain recognition. A striking relationship between binding affinity and chain length suggests that maximum affinity is associated with a conformational feature that is fully formed in chains of n = 4-6 and can be recognized by a single UBA domain of HHR23A. Our findings provide new insights into polyubiquitin chain recognition and set the stage for future structural investigations of UBA/polyubiquitin complexes.

Cross-Linking Reagents↗

PubMed2004

Sequence context modulates the stability of a GxxxG-mediated transmembrane helix-helix dimer.

To quantify the relationship between sequence and transmembrane dimer stability, a systematic mutagenesis and thermodynamic study of the protein-protein interaction residues in the glycophorin A transmembrane helix-helix dimer was carried out. The results demonstrate that the glycophorin A transmembrane sequence dimerizes when its GxxxG motif is abolished by mutation to large aliphatic residues, suggesting that the sequence encodes an intrinsic propensity to self-associate independent of a GxxxG motif. In the presence of an intact GxxxG motif, the glycophorin A dimer stability can be modulated over a span of -0.5 kcal mol(-1) to +3.2 kcal mol(-1) by mutating the surrounding sequence context. Thus, these flanking residues play an active role in determining the transmembrane dimer stability. To assess the structural consequences of the thermodynamic effects of mutations, molecular models of mutant transmembrane domains were constructed, and a structure-based parameterization of the free energy change due to mutation was carried out. The changes in association free energy for glycophorin A mutants can be explained primarily by changes in packing interactions at the protein-protein interface. The energy cost of removing favorable van der Waals interactions was found to be 0.039 kcal mol(-1) per A2 of favorable occluded surface area. The value corresponds well with estimates for mutations in bacteriorhodopsin as well as for those mutations in the interiors of soluble proteins that create packing defects.

Amino Acid Motifs↗

PubMed2004

Thermodynamics of glycophorin A transmembrane helix dimerization in C14 betaine micelles.

We have used sedimentation equilibrium analytical ultracentrifugation to measure the free energy change for the glycophorin A transmembrane helix-helix dimerization in C14 betaine micelles. By varying the amount of micellar C14 betaine, we show that the protein association reaction in the micellar C14 phase behaves as an ideal-dilute solution. In this hydrophobic environment, the mole-fraction standard state free energy change for self-association of the SNGpA99 glycophorin A construct is -5.7 (+/-0.3, N=5) kcal mol(-1) at 25 degrees C. Compared with previous results carried out in C(8)E(5) micellar solutions, the free energy of dimerization is 1.3 kcal mol(-1) less favorable in C14 betaine micelles. In contrast, when considered on a per-interface basis, the formation of the glycophorin A transmembrane dimer in C14 betaine micelles may be more favorable than the association of several designed transmembrane peptides.

Betaine↗

PubMed2002

Standardizing the free energy change of transmembrane helix-helix interactions.

Side-to-side associations of transmembrane alpha-helices are integral components of the structure and function of helical membrane proteins. A fundamental unknown in the understanding of the chemical principles driving the lateral interactions between transmembrane alpha-helices is the balance of forces arising from the polypeptide sequence versus the hydrophobic solvent. To begin to address this question, a consideration of basic thermodynamic principles has been applied to assess the experimental free energy change associated with transmembrane helix dimerization in micelles. This analysis demonstrates the ability to partition the apparent free energy of transmembrane helix-helix association into two components. The first component is a statistical energy term, which arises from the fact that there are an unequal number of reactants and products. The second component is a standard state free energy change, which informs on the molecular details of the transmembrane helix self-association reaction. The advantage of separating these two energy terms arises from the fact that extrapolation to the standard state free energy change normalizes the statistical energy term so that it applies equivalently in all experimental systems. Accompanying experimental results for the glycophorin A transmembrane alpha-helix dimer measured in micelles are well described by these theoretical components assuming an ideal-dilute solution.

Amino Acid Sequence↗