AlloRep: A Repository of Sequence, Structural and Mutagenesis Data for the LacI/GalR Transcription Regulators

Abstract

Protein families evolve functional variation by accumulating point mutations at functionally important amino acid positions. Homologs in the LacI/GalR family of transcription regulators have evolved to bind diverse DNA sequences and allosteric regulatory molecules. In addition to playing key roles in bacterial metabolism, these proteins have been widely used as a model family for benchmarking structural and functional prediction algorithms. We have collected manually curated sequence alignments for >3000 sequences, in vivo phenotypic and biochemical data for >5750 LacI/GalR mutational variants, and noncovalent residue contact networks for 65 LacI/GalR homolog structures. Using this rich data resource, we compared the noncovalent residue contact networks of the LacI/GalR subfamilies to design and experimentally validate an allosteric mutant of a synthetic LacI/GalR repressor for use in biotechnology. The AlloRep database (freely available at www.AlloRep.org) is a key resource for future evolutionary studies of LacI/GalR homologs and for benchmarking computational predictions of functional change.

Keywords: allostery; contact map; sequence alignment; transcription regulation; variant database.

Figure 1

Overview of the AlloRep database. Each structure at the top shows the two monomers of a representative LacI/GalR homodimer (pdb: 1efa; [50]) in blue and yellow, respectively. DNA is shown as a ribbon at the top of each structure. The allosteric binding sites on the regulatory domains are indicated by the presence of a black ligand. Contacts are defined when any non-hydrogen atoms of two residues come within 5 Å of each other.

Figure 2

Examples of residue contact-networks. Structures are represented as in Fig 1. For each panel, the network models are super-imposed over the space-filling model of the structure. For these contact networks, nodes are represented as the Cα atoms and the atomic contacts between two residues are represented as edges. Residues are considered to be in contact if any of their non-hydrogen atoms are within 5 Å of each other. (A) Network representation of the common noncovalent contacts that are present in all available PurR and LacI structures. (B) Network representation of the noncovalent contacts common to apo-PurR structures (no DNA bound) and LacI structures with DNA-bound. (C) Network representation of the noncovalent contacts common to LacI structures without DNA and DNA-bound-PurR conformations. Figures were prepared using Pymol [47].

Figure 3

Experimental validation of an allosteric prediction. A) Network representation of residue contacts for position 118. These networks were derived using all available, full-length structures of LacI/GalR homologs. A region of the LacI homodimer is shown, with monomers colored as in Fig 1; DNA is shown as a ribbon at the top of the structure. Position 118 (one per monomer) is located at the centers of the two networks that are super-imposed on the structure. Other contacting residues are shown as connecting nodes. B) Construction of the LLhE-3mut synthetic repressor [21]. Monomers of this chimera comprise (i) the DNA binding domain and linker of E.coli LacI, and (ii) the regulatory domain from T. fusca CelR. For measurable repression, an additional three mutations were required in the linker: I48V, Q55A, and Q60R (represented as “x”). In this study, the H118R mutation was added to LLhE-3mut to alter allosteric regulation (represented as a red dot). C) Repression of LLhE-3mut and LLhE-3mut/H118R variants as a function of inducer concentration. The repressor variants were used to control expression of green fluorescent protein (GFP). At low cellobiose concentrations, both variants repressed the gfp coding region, and GFP expression could not be detected. Increasing cellobiose concentrations induced the LLhE-3mut variants, allowing expression of GFP in a dose-dependent manner. Note that the H118R mutation required >10-fold more cellobiose, which indicates that position 118 participates in allosteric regulation. Data represent the averages of 3 or 4 independent measurements; error bars (which are usually smaller than the data symbols) show the standard deviation. Lines are to aid visual inspection of the data.