Functionally important positions can comprise the majority of a protein's architecture

Abstract

Concomitant with the genomic era, many bioinformatics programs have been developed to identify functionally important positions from sequence alignments of protein families. To evaluate these analyses, many have used the LacI/GalR family and determined whether positions predicted to be "important" are validated by published experiments. However, we previously noted that predictions do not identify all of the experimentally important positions present in the linker regions of these homologs. In an attempt to reconcile these differences, we corrected and expanded the LacI/GalR sequence set commonly-used in sequence/function analyses. Next, a variety of analyses were carried out (1) for the entire LacI/GalR sequence set and (2) for a subset of homologs with functionally-important "YxPxxxAxxL" motifs in their linkers. This strategy was devised to determine whether predictions could be improved by knowledge-based sequence sorting and -for some analyses -did increase the number of linker positions identified. However, two functionally important linker positions were not reliably identified by any analysis. Finally, we compared the new predictions to all known experimental data for E. coli LacI and three homologous linkers. From these, we estimate that >50% of positions are important to the functions of the LacI/GalR homologs. In corollary, neutral positions might occur less frequently and might be easier to detect in sequence analyses. Although analyses have successfully guided mutations that partially exchange protein functions, a better experimental understanding of the sequence/ function relationships in protein families would be helpful for uncovering the remaining rules used by nature to evolve new protein functions.

Flexibility and Disorder in Gene Regulation: LacI/GalR and Hox Proteins

“…The LacI/GalR family of transcription regulators comprises Ͼ4000 homologs; all members of this family are found exclusively in bacteria (10,12,13). The common structure of this family is a homodimer that contains one DNA-binding site and two binding sites for small-molecule, allosteric ligands (10).…”

“…1, A-C) (14,15). In ϳ60% of LacI/GalR homologs (13), this linker includes a conserved motif that forms a "hinge helix" in known structures. The side chains of the hinge helices interact with the minor groove at the center of the two DNA half-sites, bending the operator by ϳ45° (Fig.…”

“…In contrast, PurR is repressible-the consequence of binding its allosteric ligand is to enhance DNA binding and repression (15). In addition, for ϳ40% of homologs, the ϳ18-amino acid linker that connects the core domain to the DNA-binding domain appears to be completely disordered, lacking a hinge helix (13). Similar to eukaryotic intrinsically disordered proteins (26), the linker sequence in these proteins has a high density of charge and/or prolines, although the specific positions vary (Fig.…”

“…In these homologs, disorder in the linker appears to have arisen to facilitate binding DNA operators with varied spacing between half-sites ( Fig. 1E) (13,27).…”

“…Note that the hinge helix and N-terminal DNA-binding domain are not resolved in the presence of inducer, presumably due to flexibility that arises from hinge helix unfolding; the DNA-binding domain may also become less structured. D and E, linker sequence variation and DNA sites for subsets of LacI/GalR homologs that contain (top) and lack (bottom) the YPAL motif (13). In YPAL homologs, structures show that amino acids in positions Pϩ1 through Lϩ2 fold into an ␣ helix (14,15,17).…”