Plasticity of quaternary structure: Twenty‐two ways to form a LacI dimer

Abstract

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.Keywords: Structural plasticity; quaternary structure; lactose repressor protein; purine repressor protein; periplasmic binding protein; trehalose repressor protein One fascinating aspect of protein folding is that a wide range of primary sequences can generate very similar secondary and tertiary structures. For example, Thornton and her colleagues have demonstrated that many sequences with less than 35% identity have the same folding topology (Swindells et al. 1998; this reference also provides a very helpful review of the various structural classification schemes currently available). Plasticity of folding is well illustrated by the LacI/GalR family of repressor proteins. Article and publication are at www.proteinscience.org/cgi/doi/10.1110/ ps.35801. † The Escherichia coli LacI has long served as a prototype for proteins that negatively control transcription. In vivo, LacI binds tightly to a specific operator DNA site, preventing transcription of downstream lactose metabolic genes. If lactose becomes the predominant energy source, the bacteria first produce the metabolite allolactose, which binds to LacI and induces a conformational switch to a protein structure with only nonspecific DNA-binding affinity. The large excess of genomic bacterial DNA

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

“…The interface between monomers buries ∼2200 Å 2 of solvent accessible surface area, nearly equally distributed between the interfaces of the two subdomains [60,61]. Upon binding inducer, residues in the N-subdomain show significant movement with respect to their locations in the DNA-bound complex, a property that is crucial for allosteric regulation [61,[75][76][77]. In contrast, the C-subdomains remain essentially unchanged in their orientation in all liganded states (Fig.…”

“…The ability of the LacI/GalR family to form dimeric or higher-order oligomers is crucial for high specificity in DNA binding. To elucidate the evolutionary determinants of assembly, a structure-based sequence alignment of several repressors and periplasmic binding proteins and a series of contact maps in network representation were generated for the repressor interfaces [76,77]. These analyses revealed that the primary sequences corresponding to the interfaces of LacI/GalR family C-subdomains differ significantly from the monomeric PBPs in the region around LacI residues 281 and 282, sites in LacI known to be important in assembly.…”

“…In vitro characterization of this monomer variant demonstrated that the protein was folded with inducer binding properties comparable to the wild-type repressor [120]. Interestingly, dimerization of the Y282D variant could be compensated by a secondsite mutation (K84L or K84A) in the N-subdomain [108], and 22 additional compensatory second-site mutations were identified by phenotypic screening for variants with restored repression function [76]. The large number and diversity of second site revertants suggests that oligomerization of the PBP-like protein (LacI monomer) is robust, with many different scenarios for assembly.…”

Engineering Soluble Monomeric Streptavidin with Reversible Biotin Binding Capability

“…To engineer monomeric streptavidin with a minimal number of mutated residues, an attractive approach is to introduce both charge repulsion and steric hindrance at these interfaces. As protein has structural plasticity (23)(24)(25), it is vital to select interfacial residues located on a rigid surface to maximize the effects of charge repulsion and steric hindrance. Because streptavidin subunit forms an eightantiparallel stranded ␤-barrel structure (4, 5), the selected …”

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

“…In this network, amino acid positions are represented as nodes and their non-covalent interactions with other positions comprise the network edges (Fig 1); the latter are delimited by distance thresholds (here, 5 Å between the two closest atoms in the PDB structure). This representation retains information about individual atoms, reduces the visual complexity, links protein structural interpretation with graph theory [35], and allows a parallel analysis of multiple structures [34,36]. We previously used this approach for comparing protein-protein interfaces [32,33,36,37].…”