Modeling the Molecular Evolution of Protein Domains and Networks.

MCFERRIN, LISA GAIL. Modeling the Molecular Evolution of Protein Domains and Networks. (Under the direction of William R. Atchley and Eric Stone.) Protein sequences are subject to a mosaic of constraints. Such constraints often manifest in patterns of conservation and can reveal important structural or functional features among homologous sequences. Regions of constraint on the tertiary structure of a protein result in loose segmentation of its primary structure into stretches of slowlyand rapidly-evolving amino acids. We demonstrate that the regional nature of structural and functional constraints asserts a positive autocorrelation on the evolutionary rates of neighboring sites. Using a simple dispersion statistic that quantifies the degree of non-synonymous clustering for genome-wide interspecific comparisons of orthologous protein pairs, we show non-synonymous clustering intensifies with increasing purifying selection, revealing a strong log-linear relationship between the degree of clustering and the intensity of constraint. This relationship is also preserved in yeast, even after accounting for other selection correlates such as protein abundance and dispensability. While we do not claim that the dispersion ratio is an optimal statistic, we propose that it may help uncover conflicting signals of constraint that would otherwise be lost in a historical switch in selective regime. In general, the correlation between clustering and evolutionary rate supports the use of de novo annotation methods that have implicitly assumed these selective constraints. Regional constraint also forms conserved domains such as the basic-helix-loop-helixzipper (bHLHZ) domain responsible for DNA binding and dimerization. The bHLHZ domain defines the interactions between Max and Mlx transcription factor network members (Max, Mlx, Mondo, Mga, Mnt, Mxd, and Myc), which are integral for differentiation, proliferation, and energy metabolism. We identified the bHLHZ domain of Max and Mlx network members in all sampled animal lineages, dating these networks to ! 1 billion years ago. Throughout evolution, only four major network configurations emerged 1) most species contain core network members (Myc, Max, Mlx, Mondo, Mnt, and Mxd), 2) vertebrates experienced additional radiation of protein families (c-, N-, and L-Myc, Mxd1-4, MondoA and MondoB), 3) Mxd was lost in the Diptera lineage, and 4) a major network reconstruction in nematodes formed a Mxd-like (MDL-1), a Myc and Mondo-like (MML-1), a Mlx-like (Mxl-2), and two Max-like (Mxl-1, Mxl-2) proteins. Phylogenetic reconstruction of the bHHZ domain shows distinct conservation among orthologous proteins, while multivariate discriminant analysis reveals particular residues that classify proteins, families, and network configurations. Such differences likely affect the recognition of gene target sequences, and hence alter the function of these transcription factors. In vertebrates, MondoA and MondoB paralogs regulate energy homeostasis by transactivating genes involved in glucose metabolism. In addition to the bHLHZ domain, five unique N-terminus domains named Mondo Conserved Regions (MCRI-V) regulate their transactivational activity in response to glucose-6-phosphate (G6P). Through sequence conservation, we identified an additional constrained region (MCR6) that is located in a region capable of transactivation. MCR6 has a SxSxxT motif that is similar to known G6P binding regions, suggesting G6P may bind to MCR6 allosterically. MCR6 also matches the sequence signature of a 9aa transactivation domain known to interact with histone acetyltransferases such as MondoB interaction partner CBP/p300. Structural predictions of the N-terminal region agree with existing experimental evidence, which indicate that intramolecular interactions between MCRI-IV, MCR6 and MCRV may affect MondoA and MondoB conformation in different glucose conditions. Hence, MCR6 may provide the necessary link in determining how MondoA and MondoB are able to transactivate genes in response to glucose. Modeling the Molecular Evolution of Protein Domains and Networks by Lisa Gail McFerrin A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy