Functional consequences of exchanging domains between LacI and PurR are mediated by the intervening linker sequence

Abstract

Homologue function can be differentiated by changing residues that affect binding sites or long-range interactions. LacI and PurR are two proteins that represent the LacI/GalR family (>500 members) of bacterial transcription regulators. All members have distinct DNA-binding and regulatory domains linked by approximately 18 amino acids. Each homologue has specificity for different DNA and regulatory effector ligands; LacI and PurR also exhibit differences in allosteric communication between DNA and effector binding sites. A comparative study of LacI and PurR suggested that alterations in the interface between the regulatory domain and linker are important for differentiating their functions. Four residues (equivalent to LacI positions 48, 55, 58, and 61) appear particularly important for creating a unique interface and were predicted to be necessary for allosteric regulation. However, nearby residues in the linker interact with DNA ligand. Thus, differences observed in interactions between linker and regulatory domain may be the cause of altered function or an effect of the two proteins binding different DNA ligands. To separate these possibilities, we created a chimeric protein with the LacI DNA-binding domain/linker and the PurR regulatory domain (LLhP). If the interface requires homologue-specific interactions in order to propagate the signal from effector binding, then LLhP repression should not be allosterically regulated by effector binding. Experiments show that LLhP is capable of repression from lacO1 and, contrary to expectation, allosteric response is intact. Further, restoring the potential for PurR-like interactions via substitutions in the LLhP linker tends to diminish repression. These effects are especially pronounced for residues 58 and 61. Clearly, binding affinity of LLhP for the lacO1 DNA site is sensitive to long-range changes in the linker. This result also raises the possibility that mutations at positions 58 and 61 co-evolved with changes in the DNA-binding site. In addition, repression measured in the absence and presence of effector ligand shows that allosteric response increases for several LLhP variants with substitutions at positions 48 and 55. Thus, while side chain variation at these sites does not generally dictate the presence or absence of allostery, the nature of the amino acid can modulate the response to effector.

Comparing the Functional Roles of Nonconserved Sequence Positions in Homologous Transcription Repressors: Implications for Sequence/Function Analyses

“…For example, LLhP (a chimera with the LacI DNA-binding domain and linker and the PurR regulatory domain), is co-repressed; whereas the LLhG chimera with the GalR regulatory domain is induced, each by their respective ligands 19; 21. In this study, we expected and observed similar effector responses for LPhP and LGhG chimeras.…”

“…LPhP and LGhG were constructed in order to compare the functional contributions of PurR and GalR linker positions to those in the LacI linkers in LLhP and LLhG19; 21. In Materials and Methods, we present our criteria for designing the chimeric repressors and for interpreting in vivo repression resultsb.…”

“…Previously, we reported experimental results for two chimeras comprising the LacI DNA-binding domain, the LacI linker, and either the PurR or the GalR regulatory domain (respectively named LLhP and LLhG 19; 21, Figure 1C). Using a “host-guest” strategy, we substituted up to thirteen amino acids into several nonconserved linker positions, which resulted in a range of functional changes.…”

Ligand-induced Conformational Changes and Conformational Dynamics in the Solution Structure of the Lactose Repressor Protein

“…Such a model is consistent with mutagenesis of an engineered homologue, which showed that linker interface mutations can alter repression of either or both the low-and high-affinity complexes. 56 The ternary oDNA complex must occur in vivo, but is short-lived, since excess genomic DNA effectively competes to displace the repressor protein from the operator. [16][17][18] Therefore, another question is whether the structure of LacI in the oDNA ternary complex is distinct from the repressor structure in complex with nDNA.…”

Mathematical modeling of the apo and holo transcriptional regulation in Escherichia coli

“…In mathematical terms, this implies an additive effect of o and r over the mutation rates, c, i 1 , j 2 , and k 2 , (Table S1, ESI †). This intrinsic TF interaction has been experimentally reported, at least for the well-documented LacI, by molecular structure analysis, 15 and by changing residues that affect the binding site, 16 among others.…”