Edwin Li

Transmembrane helix dimerization: beyond the search for sequence motifs.

Studies of the dimerization of transmembrane (TM) helices have been ongoing for many years now, and have provided clues to the fundamental principles behind membrane protein (MP) folding. Our understanding of TM helix dimerization has been dominated by the idea that sequence motifs, simple recognizable amino acid sequences that drive lateral interaction, can be used to explain and predict the lateral interactions between TM helices in membrane proteins. But as more and more unique interacting helices are characterized, it is becoming clear that the sequence motif paradigm is incomplete. Experimental evidence suggests that the search for sequence motifs, as mediators of TM helix dimerization, cannot solve the membrane protein folding problem alone. Here we review the current understanding in the field, as it has evolved from the paradigm of sequence motifs into a view in which the interactions between TM helices are much more complex. This article is part of a Special Issue entitled: Membrane protein structure and function.

Receptor tyrosine kinase transmembrane domains: Function, dimer structure and dimerization energetics

The transmembrane (TM) domains of receptor tyrosine kinases (RTKs) play an active role in signaling. They contribute to the stability of full-length receptor dimers and to maintaining a signaling-competent dimeric receptor conformation. In an exciting new development, two structures of RTK TM domains have been solved, a break-through achievement in the field. Here we review these structures, and we discuss recent studies of RTK TM domain dimerization energetics, possible synergies between domains, and the effects of pathogenic RTK TM mutations on structure and dimerization.

Application of temperature control strategies to the growth of hen egg-white lysozyme crystals

Abstract Solubility data were combined with mass balances and growth kinetics to derive a temperature control algorithm which maintains a constant level of supersaturation. This constant supersaturation control (CSC) algorithm attempts to maximize the size of protein crystals by maintaining the growth conditions in the metastable zone. Using hen egg-white lysozyme as a model protein system, four temperature programming strategies were employed in seeded and unseeded systems: the CSC algorithm, a linear ramp derived from the CSC algorithm, isothermal 20°C, and isothermal 4°C. Both the CSC-derived linear and the CSC temperature programs yielded large, well-formed crystals which were significantly larger than crystals grown isothermally at 20 and 4°C. The isothermal 4°C program resulted in poorly formed crystals due to the high initial growth rates. The seeded systems displayed much higher levels of nucleation than the unseeded systems which is attributed to secondary nucleation. The results indicate that moderate deviations (∼ 20%) from constant supersaturation can be tolerated, while still producing large, well-formed crystals appropriate for X-ray crystallography.

Assembly of the M2 Tetramer Is Strongly Modulated by Lipid Chain Length

The influenza virus matrix protein 2 (M2) assembles into a tetramer in the host membrane during viral uncoating and maturation. It has been used as a model system to understand the relative contributions of protein-lipid and protein-protein interactions to membrane protein structure and association. Here we investigate the effect of lipid chain length on the association of the M2 transmembrane domain into tetramers using Förster resonance energy transfer. We observe that the interactions between the M2 helices are much stronger in 1,2-dilauroyl-sn-glycero-3-phosphocholine than in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. Thus, lipid chain length and bilayer thickness not only modulate peptide interactions, but could also be a major determinant of the association of transmembrane helices into functional membrane protein oligomers.

Increased expression of the integral membrane protein ErbB2 in Chinese hamster ovary cells expressing the anti-apoptotic gene Bcl-xL

Receptor tyrosine kinases (RTKs) are the second largest family of membrane receptors and play a key role in the regulation of vital cellular processes, such as control of cell growth, differentiation, metabolism, and migration. The production of whole-length RTKs in large quantities for biophysical or structural characterization, however, is a challenge. In this study, a cell engineering strategy using the anti-apoptotic Bcl-2 family protein, Bcl-x(L), was tested as a potential method for increasing stable expression levels of a recombinant RTK membrane protein in Chinese hamster ovary (CHO) cells. Wild-type and CHO cells stably overexpressing heterologous Bcl-x(L) were transformed with the gene for a model RTK membrane protein, ErbB2, on a plasmid also containing the Zeocin resistance gene. While CHO cells exhibited a gradual decrease in expression with passaging, CHO-bcl-x(L) cells offered an increased and sustained level of ErbB2 expression following continuous passaging over more than 33 days in culture. The increased ErbB2 expression in CHO-bcl-x(L) cells was evident both in stable transfected pools and in clonal isolates, and demonstrated both in Western blot analysis and flow cytometry. Furthermore, the sustained high-level protein expression in CHO-bcl-x(L) cells does not alter the correct membrane localization of the ErbB2 protein. Our results demonstrate that cellular engineering, specifically anti-apoptosis engineering, can provide increased and stable ErbB2 membrane protein expression in mammalian cells. This approach may also be useful for other membrane proteins in which large quantities are needed for biophysical and structural studies.

Utility of surface-supported bilayers in studies of transmembrane helix dimerization

This review focuses on the methods that are available to study transmembrane (TM) helix dimerization in membrane-like environments (either bacterial membranes or lipid bilayers, as mimics of the eukaryotic cellular membrane), with an emphasis on the utility of surface-supported bilayers in such studies.

Increased expression of the integral membrane proteins EGFR and FGFR3 in anti-apoptotic Chinese hamster ovary cell lines

Membrane proteins such as receptor tyrosine kinases (RTKs) have a vital role in many cellular functions, making them potential targets for therapeutic research. In this study, we investigated the coexpression of the anti‐apoptosis gene Bcl‐xL with model membrane proteins as a means of increasing membrane protein expression in mammalian cells. Chinese hamster ovary (CHO) cells expressing heterologous Bcl‐xL and wild‐type CHO cells were transfected with either epidermal growth factor receptor or fibroblast growth factor receptor 3. The CHO–Bcl‐xL cell lines showed increased expression of both RTK proteins as compared with the wild‐type CHO cell lines in transient expression analysis, as detected by Western blot and flow cytometry after 15 days of antibiotic selection in stable expression pools. Increased expression was also seen in clonal isolates from the CHO–Bcl‐xL cell lines, whereas the clonal cell line expression was minimal in wild‐type CHO cell lines. Our results demonstrate that application of the anti‐apoptosis gene Bcl‐xL can increase expression of RTK proteins in CHO cells. This approach may be applied to improve stable expression of other membrane proteins in the future using mammalian cell lines with Bcl‐xL or perhaps other anti‐apoptotic genes.

Surface supported bilayer platform for studies of lateral association of proteins in membranes (Mini Review)

Here, the authors review how surface supported bilayers can be engineered and how Förster resonance energy transfer (FRET) can be used to quantify interactions between transmembrane peptides in these bilayers. The requirements for the surface supported platform are (1) lateral mobility of the peptides, (2) transmembrane orientation of the peptides, and (3) capabilities for FRET measurements. To satisfy these requirements, a new assembly method, termed “directed assembly” was developed. This assembly method could have broad utility in basic studies of proteins in membranes and in biotechnological applications.

The Pathogenic A391E Mutation in FGFR3 Induces a Structural Change in the Transmembrane Domain Dimer

Fibroblast growth factor receptor 3 (FGFR3) is a single-pass membrane protein and a member of the receptor tyrosine kinase family of proteins that is involved in the regulation of skeletal growth and development. FGFR3 has three distinct domains: the ligand binding extracellular domain, the cytosolic kinase domain and the transmembrane domain (TMD). Previous work with the isolated FGFR3 TMD has shown that it has the ability to dimerize. Clinical and genetic studies have also correlated mutations in the TMD with a variety of skeletal and cranial dysplasias and cancer. Although the structures of the extracellular and cytosolic domains of FGFR3 have been solved, the structure of the TMD dimer is still unknown. Furthermore, very little is known regarding the effects of pathogenic mutations on the TMD dimer structure. We, therefore, carried out ToxR activity assays to determine the role of the SmXXXSm motif in the dimerization of the FGFR3 TMD. This motif has been shown to drive the association of many transmembrane proteins. Our results indicate that the interaction between wild-type FGFR3 TMDs is not mediated by two adjacent SmXXXSm motifs. In contrast, studies using the TMD carrying the pathogenic A391E mutation suggest that the motifs play a role in the dimerization of the mutant TMD. Based on these observations, here we report a new mechanistic model in which the pathogenic A391E mutation induces a structural change that leads to the formation of a more stable dimer.

The Role of Two SmXXXSm Motifs in the Dimerization of the FGFR3 Transmembrane Domain

Fibroblast growth factor receptor 3 (FGFR3) is a single pass membrane protein and a member of the receptor tyrosine kinase (RTK) family of proteins. FGFR3 has three distinct domains: the ligand binding extracellular domain, the cytosolic kinase domain, and the transmembrane (TM) domain. Previous work with the isolated FGFR3 TM domains has shown that it has the ability to self-dimerize. The TM domain of FGFR3 has two adjacent SmXXXSm motifs in the sequence, where the Sm residues are Ala374, Ser378 and Gly382. The SmXXXSm motif has been shown to play an important role in helix- helix interaction in other transmembrane proteins such as GpA and BNIP3. Currently, we are using site directed mutagenesis and ToxR activity assays to analyze the importance of this sequence motif for the interaction between the helices. Single mutations were made at three specific points, changing the small amino acids (Ala374, Ser378, and Gly382) to five different amino acids (Ala, Cys, Gly, Ile, and Ser). Relative activity measurements were made comparing the activity of the mutant protein to that of the wild type protein. Through these comparative measurements we can assess their role in helix-helix interaction.