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Implementing a DNA Library to Explore the Sequence Dependence of Dimerization of BNIP3-like Transmembrane Domains

by: Kushagra Shrinath

For the past decade here at Rice, the MacKenzie lab has been studying transmembrane proteins (proteins that span a biological membrane) and the self-association of alpha-helices within these transmembrane proteins. Standard biophysical and biochemical assays are carried out on point mutants of important transmembrane proteins such as BNIP3 (a protein that promotes apoptosis), Syndecan (a receptor protein linked to various growth factors), and Glycophorin A (a protein that spans the membrane of a red blood cell). By assaying how different point mutants affect the dimerization of the transmembrane proteins, it is possible to deduce both the importance of the location of residues to dimerization, as well as how different residues at particular locations affect dimerization. Due to the critical role that transmembrane domains play in cellular signaling, figuring out motifs that cause dimerization is an important first step to help regulate abnormal cell signaling which can lead to metabolic and immunological disorders, as well as cancer. In this paper we will discuss transmembrane proteins, measures of studying them biochemically, and recent studies in the MacKenzie lab involving DNA libraries.

Transmembrane proteins are integral in the structure and function of cells. They constitute 27% of all proteins made by humans1 and serve a variety of functions in cell signaling and regulating proper ion concentrations. Although very important in biology, transmembrane proteins are not simple to study. Their helical structure, and moreover the helix-helix interactions caused by this structure, is a result of the amphiphilic (polar and non-polar) environment in which they reside. This amphiphilic environment makes biophysical and biochemical studies of transmembrane proteins difficult.5 Moreover, the accuracy of efforts to use detergent micelles to simulate the environment is unknown.

An assay called TOXCAT established by Russ and Engelman however, allows for in vivo measurements of an important facet of transmembrane domains: their ability to dimerize.6 By fusing ToxR, a transcription factor dependent on dimerization, to the transmembrane domain, one is able to detect dimerization when ToxR activates a reporter gene encoding for chloramphenicol acetyl transferase (CAT) via the cholera toxin promoter (ctx). (Fig. 1). Russ and Engelman used TOXCAT to measure the effect of single point mutations on Glycophorin A and found that mutations to polar residues show specificity in TOXCAT while they are disruptive in SDS.

The ability of a transmembrane domain to dimerize is largely dependent on the sequence of the interfacial residues that are active in dimerization.4 While point mutants are helpful in deducing how single residue changes affect dimerization, they are impractical to use when trying to uncover motifs and general patterns that cause tight dimers. The number of possible mutants that can be made is astronomically large, especially when considering double and triple mutants. Furthermore, the vast majority of these mutants are not strongly dimerizing and it would not be viable to use resources to carry out assays on them. What is needed is an assay that produces numerous mutants and also selects for mutants that are strongly dimerizing. It is here where a DNA library is a valuable tool to explore the sequence dependent dimerization of transmembrane proteins. Whereas mutagenesis alters a sequence at one position, a DNA library contains several degenerate nucleotides at key positions of interest in the transmembrane protein, thereby allowing it encode for many mutants while still maintaining the overall structure of the protein the same. Furthermore, single point mutations also ignore residues that work in tandems or combinations. Instead of focusing on residues at single locations, a library allows motifs of residues to be uncovered. By using a library it is also possible to define select residues at the interfacial region and allow for cut sites that eliminate certain residues altogether. This project aims to assess the general framework that is necessary for dimerization via the use of a DNA library. This library codes for human BNIP3 which has been integral in apoptosis2 as well as C. elegans BNIP3, Glycophorin A, and Syndecan 3.

Synthetic oligonucleotides that have the same backbone but encode for different chosen amino acids at the interfacial positions of the transmembrane domain will be amplified by PCR. They will be inserted into a pccKan vector in between the N-terminal ToxR transcription factor and the C-terminal Maltose Binding Protein. The ligated vector will be transformed into E. coli and the DNA harvested will represent the library from which future screening will take place. This DNA will then be transformed into NT326 cells which allow for transmembrane domains that dimerize strongly to be resistant to chloramphenicol. By growing up the NT326 cells on increasing concentrations of chloramphenicol and sequencing a number of cells that survive at various concentrations, one can deduce the residues and motifs that favor dimerization. Finally, the sequences of interest can be assayed via TOXCAT and their relative dimerization values can be compared. Through many screenings, a large enough library can be constructed that will allow for one to see the effect of an amino acid at a single position on the transmembrane domain. In addition, comprehensive motifs that tightly dimerize will be found.

One feature that has been seen by Russ et. al. is the predominance of a GxxxG motif in dimerization.7 In the absence of a GxxxG motif, a library implemented by Dawson et al. showed that the most tightly dimerizing sequences had polar serines and threonines which are thought to contribute to dimerization through hydrogen bonds.3 This library is distinctive in that it allows both polar and non-polar residues to lie at interfacial residues to assess whether or not hydrogen bonding is in fact responsible for the tight dimerization seen in polar residues. Furthermore, this library allows the central glycine to be eliminated to uncover other tightly dimerizing motifs that lack a GxxxG motif. Results of a preliminary study involving this DNA library have shown that on the most tightly dimerizing transmembrane protein, there is always a glycine present at the central position and a strong, statistically significant presence of phenylalanine 3 positions prior. Polar residues (serine and threonine) are prevalent at the first position of transmembrane domain, while the branched amino acids leucine and valine predominate at the sixth position. These findings provide insight to general motifs that arise as a result of induced variation and selection and give us the ability to predict the dimerization strengths of similar transmembrane sequences found in biological systems. Future work will include expanding the DNA library as well as creating a new library to ask different questions by changing the residues that can be at a particular position.

The use of a DNA library is an elegant method with which important questions can be answered in a holistic and encompassing manner without expending a lot of resources. Its application with transmembrane domains will potentially reveal important motifs that lead to strong dimerization. This information can be used to make tailor-designed drugs or novel mechanisms that can disrupt or enhance the dimerization of biological transmembrane proteins. Due to the large amount of biological mechanisms regulated by the self-association of alpha-helices, this information can be vital in designing a method to treat a myriad of disorders.

References

1. Almén, M.S., Nordström, K.J., Fredriksson, R., Schiöth, H.B. Mapping the human membrane proteome: a majority of the human membrane proteins can be classified according to function and evolutionary origin. BMC Biology 2009, 7:50.
2. Chen, G., Ray, R., Dubik, D., Shi, L., Cizeau, J., Bleackl, R.C., Saxena, S., Gietz, R.D., and Greenberg, A.H. The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis. Journal of Experimental Medicine 1997, 186, 1975-1983.
3. Dawson, J.P., Weinger, D.S., Engelman, D.M. Motifs of serine and threonine can drive association of transmembrane helices. Journal of Molecular Biology 2002, 316, 799-805.
4. Lemmon ,M.A., Flanagan, J.M., Treutlein, H.R., Zhang, J, Engelman, D.M. Sequence specificity in the dimerization of transmembrane helices. Biochemistry 1992, Dec 29;31(51):12719-25.
5. MacKenzie, K.R. Folding and Stability of α-Helical Integral Membrane Proteins. Journal of Biological Chemistry 2006, 106, 1931-1977.
6. Russ, W.P., and Engelman, D.M. TOXCAT: A measure of transmembrane helix association in a biological membrane. Proceedings of the National Academy of Science 1999, 96, 863-868.
7. Russ, W.P, and Engelman, D.M. The GxxxG motif: a framework for transmembrane helix-helix association. Journal of Molecular Biology 2000, 296, 911-919.

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