Liskin Swint-Kruse

The lactose repressor system: paradigms for regulation, allosteric behavior and protein folding

Abstract.In 1961, Jacob and Monod proposed the operon model for gene regulation based on metabolism of lactose in Escherichia coli [1]. This proposal was followed by an explication of allosteric behavior by Monod and colleagues [2]. The operon model rationally depicted how genetic mechanisms can control metabolic events in response to environmental stimuli via coordinated transcription of a set of genes with related function (e.g. metabolism of lactose). The allosteric response found in the lactose repressor and many other proteins has been extended to a variety of cellular signaling pathways in all organisms. These two models have shaped our view of modern molecular biology and captivated the attention of a surprisingly broad range of scientists. More recently, the lactose repressor monomer was used as a model system for experimental and theoretical explorations of protein folding mechanisms. Thus, the lac system continues to advance our molecular understanding of genetic control and the relationship between sequence, structure and function.

Allostery in the LacI/GalR family: variations on a theme

The lactose repressor protein (LacI) was among the very first genetic regulatory proteins discovered, and more than 1000 members of the bacterial LacI/GalR family are now identified. LacI has been the prototype for understanding how transcription is controlled using small metabolites to modulate protein association with specific DNA sites. This understanding has been greatly expanded by the study of other LacI/GalR homologues. A general picture emerges in which the conserved fold provides a scaffold for multiple types of interactions - including oligomerization, small molecule binding, and protein-protein binding - that in turn influence target DNA binding and thereby regulate mRNA production. Although many different functions have evolved from this basic scaffold, each homologue retains functional flexibility: For the same protein, different small molecules can have disparate impact on DNA binding and hence transcriptional outcome. In turn, binding to alternative DNA sequences may impact the degree of allosteric response. Thus, this family exhibits a symphony of variations by which transcriptional control is achieved.

Plasticity of quaternary structure: twenty-two ways to form a LacI dimer.

The repressor proteins of the LacI/GalR family exhibit significant similarity in their secondary and tertiary structures despite less than 35% identity in their primary sequences. Furthermore, the core domains of these oligomeric repressors, which mediate dimerization, are homologous with the monomeric periplasmic binding proteins, extending the issue of plasticity to quaternary structure. To elucidate the determinants of assembly, a structure‐based alignment has been created for three repressors and four periplasmic binding proteins. Contact maps have also been constructed for the three repressor interfaces to distinguish any conserved interactions. These analyses show few strict requirements for assembly of the core N‐subdomain interface. The interfaces of repressor core C‐subdomains are well conserved at the structural level, and their primary sequences differ significantly from the monomeric periplasmic binding proteins at positions equivalent to LacI 281 and 282. However, previous biochemical and phenotypic analyses indicate that LacI tolerates many mutations at 281. Mutations at LacI 282 were shown to abrogate assembly, but for Y282D this could be compensated by a second‐site mutation in the core N‐subdomain at K84 to L or A. Using the link between LacI assembly and function, we have further identified 22 second‐site mutations that compensate the Y282D dimerization defect in vivo. The sites of these mutations fall into several structural regions, each of which may influence assembly by a different mechanism. Thus, the 360‐amino acid scaffold of LacI allows plasticity of its quaternary structure. The periplasmic binding proteins may require only minimal changes to facilitate oligomerization similar to the repressor proteins.

Resmap: automated representation of macromolecular interfaces as two-dimensional networks

UNLABELLED To aid detailed comparison of a large number of macromolecular structures, Resmap imports Protein Data Bank files and represents subunit/domain interfaces as two-dimensional networks. AVAILABILITY http://www.kumc.edu/biochemistry/resmap/. SUPPLEMENTARY INFORMATION Default definitions and directions for graphically managing networks are available at the same website.

Modular, multi-input transcriptional logic gating with orthogonal LacI/GalR family chimeras.

In prokaryotes, the construction of synthetic, multi-input promoters is constrained by the number of transcription factors that can simultaneously regulate a single promoter. This fundamental engineering constraint is an obstacle to synthetic biologists because it limits the computational capacity of engineered gene circuits. Here, we demonstrate that complex multi-input transcriptional logic gating can be achieved through the use of ligand-inducible chimeric transcription factors assembled from the LacI/GalR family. These modular chimeras each contain a ligand-binding domain and a DNA-binding domain, both of which are chosen from a library of possibilities. When two or more chimeras have the same DNA-binding domain, they independently and simultaneously regulate any promoter containing the appropriate operator site. In this manner, simple transcriptional AND gating is possible through the combination of two chimeras, and multiple-input AND gating is possible with the simultaneous use of three or even four chimeras. Furthermore, we demonstrate that orthogonal DNA-binding domains and their cognate operators allow the coexpression of multiple, orthogonal AND gates. Altogether, this work provides synthetic biologists with novel, ligand-inducible logic gates and greatly expands the possibilities for engineering complex synthetic gene circuits.

Fine‐tuning function: Correlation of hinge domain interactions with functional distinctions between LacI and PurR

LacI and PurR are highly homologous proteins. Their functional units are homodimers, with an N‐terminal DNA binding domain that comprises the helix‐turn‐helix (HTH), N‐linker, and hinge regions from both monomers. Hinge structural changes are known to occur upon DNA dissociation but are difficult to monitor experimentally. The initial steps of hinge unfolding were therefore examined using molecular dynamics simulations, utilizing a truncated, chimeric protein comprising the LacI HTH/N‐linker and PurR hinge. A terminal Gly‐Cys‐Gly was added to allow “dimerization” through disulfide bond formation. Simulations indicate that differences in LacI and PurR hinge primary sequence affect the quaternary structure of the hinge•hinge′ interface. However, these alternate hinge orientations would be sterically restricted by the core domain. These results prompted detailed comparison of recently available DNA‐bound structures for LacI and truncated LacI(1–62) with the PurR structure. Examination revealed that different N‐linker and hinge contacts to the core domain of the partner monomer (which binds effector molecule) affect the juxtapositions of the HTH, N‐linker, and hinge regions in the DNA binding domain. In addition, the two full‐length repressors exhibit significant differences in the interactions between the core and the C‐linker connection to the DNA binding domain. Both linkers and the hinge have been implicated in the allosteric response of these repressors. Intriguingly, one functional difference between these two proteins is that they exhibit opposite allosteric response to effector. Simulations and observed structural distinctions are correlated with mutational analysis and sequence information from the LacI/GalR family to formulate a mechanism for fine‐tuning individual repressor function.

Functional Consequences of Exchanging Domains Between LacI and PurR are Mediated by the Intervening Linker Sequence

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 ∼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. Proteins 2007.

Experimental identification of specificity determinants in the domain linker of a LacI/GalR protein: Bioinformatics-based predictions generate true positives and false negatives

In protein families, conserved residues often contribute to a common general function, such as DNA‐binding. However, unique attributes for each homolog (e.g. recognition of alternative DNA sequences) must arise from variation in other functionally‐important positions. The locations of these “specificity determinant” positions are obscured amongst the background of varied residues that do not make significant contributions to either structure or function. To isolate specificity determinants, a number of bioinformatics algorithms have been developed. When applied to the LacI/GalR family of transcription regulators, several specificity determinants are predicted in the 18 amino acids that link the DNA‐binding and regulatory domains. However, results from alternative algorithms are only in partial agreement with each other. Here, we experimentally evaluate these predictions using an engineered repressor comprising the LacI DNA‐binding domain, the LacI linker, and the GalR regulatory domain (LLhG). “Wild‐type” LLhG has altered DNA specificity and weaker lacO1 repression compared to LacI or a similar LacI:PurR chimera. Next, predictions of linker specificity determinants were tested, using amino acid substitution and in vivo repression assays to assess functional change. In LLhG, all predicted sites are specificity determinants, as well as three sites not predicted by any algorithm. Strategies are suggested for diminishing the number of false negative predictions. Finally, individual substitutions at LLhG specificity determinants exhibited a broad range of functional changes that are not predicted by bioinformatics algorithms. Results suggest that some variants have altered affinity for DNA, some have altered allosteric response, and some appear to have changed specificity for alternative DNA ligands. Proteins 2008.

Designed Disulfide between N-terminal Domains of Lactose Repressor Disrupts Allosteric Linkage

Substitution of Cys for Val at position 52 of thelac repressor was designed to permit disulfide bond formation between the two N-terminal DNA binding domains that comprise an operator DNA binding site. This position marks the closest approach of these domains based on the x-ray crystallographic structures of the homologous purine holorepressor-operator complex and lacrepressor-operator complex (Schumacher, M. A., Choi, K. Y., Zalkin, H., and Brennan, R. G. (1994) Science 266, 763–770; Lewis, M., Chang, G., Horton, N.C., Kercher, M. A., Pace, H. C., Schumacher, M. A., Brennan, R. G., and Lu, P. (1996) Science 271, 1247–1254). The V52C mutation was generated by site-specific methods, and the mutant protein was purified and characterized. In the reduced form, V52C bound operator DNA with slightly increased affinity. Exposure to oxidizing conditions resulted in disulfide bond formation, and the oxidized protein bound operator DNA with ∼6-fold higher affinity than wild-type protein. Inducer binding for both oxidized and reduced forms of V52C was comparable to wild-type lac repressor. In the presence of inducer, the reduced protein exhibited wild-type, diminished DNA binding. In contrast, DNA binding for the oxidized form was unaffected by inducer, even at 1 mm. Thus, the formation of the designed disulfide between Cys52 side chains within each dimer renders the protein-operator complex unresponsive to sugar binding, presumably by disrupting the allosteric linkage between operator and inducer binding.

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

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. Proteins 2011;