首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
Many mutations that impact protein function occur at residues that do not directly contact ligand. To understand the functional contributions from the sequence that links the DNA-binding and regulatory domains of the LacI/GalR homologues, we have created a chimeric protein (LLhP), which comprises the LacI DNA-binding domain, the LacI linker, and the PurR regulatory domain. Although DNA binding site residues are identical in LLhP and LacI, thermodynamic measurements of DNA binding affinity show that LLhP does not discriminate between alternative DNA ligands as well as LacI. In addition, small-angle scattering experiments show that LLhP is more compact than LacI. When DNA is released, LacI shows a 20 A increase in length that was previously attributed to unfolding of the linker. This change is not seen in apo-LLhP, even though the linker sequences of the two proteins are identical. Together, results indicate that long-range functional and structural changes are propagated across the interface that forms between the linker and regulatory domain. These changes could be mediated via the side chains of several linker residues that contact the regulatory domains of the naturally occurring proteins, LacI and PurR. Substitution of these residues in LLhP leads to a range of functional effects. Four variants exhibit altered affinity for DNA, with no changes in selectivity or allosteric response. Another two result in proteins that bind operator DNA with very low affinity and no allosteric response, similar to LacI binding nonspecific DNA sequences. Two more substitutions simultaneously diminish affinity, enhance allostery, and profoundly alter DNA ligand selectivity. Thus, positions within the linker can be varied to modulate different aspects of repressor function.  相似文献   

2.
3.
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 x 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.  相似文献   

4.
The crystal structure of the Escherichia coli trehalose repressor (TreR) in a complex with its inducer trehalose-6-phosphate was determined by the method of multiple isomorphous replacement (MIR) at 2.5 A resolution, followed by the structure determination of TreR in a complex with its noninducer trehalose at 3.1 A resolution. The model consists of residues 61 to 315 comprising the effector binding domain, which forms a dimer as in other members of the LacI family. This domain is composed of two similar subdomains each consisting of a central beta-sheet sandwiched between alpha-helices. The effector binding pocket is at the interface of these subdomains. In spite of different physiological functions, the crystal structures of the two complexes of TreR turned out to be virtually identical to each other with the conformation being similar to those of the effector binding domains of the LacI and PurR in complex with their effector molecules. According to the crystal structure, the noninducer trehalose binds to a similar site as the trehalose portion of trehalose-6-phosphate. The binding affinity for the former is lower than for the latter. The noninducer trehalose thus binds competitively to the repressor. Unlike the phosphorylated inducer molecule, it is incapable of blocking the binding of the repressor headpiece to its operator DNA. The ratio of the concentrations of trehalose-6-phosphate and trehalose thus is used to switch between the two alternative metabolic uses of trehalose as an osmoprotectant and as a carbon source.  相似文献   

5.
The bacterial LacI/GalR family repressors such as lactose operon repressor (LacI), purine nucleotide synthesis repressor (PurR), and trehalose operon repressor (TreR) consist of not only the N-terminal helix-turn-helix DNA-binding domain but also the C-terminal ligand-binding domain that is structurally homologous to periplasmic sugar-binding proteins. These structural features imply that the repressor family evolved by acquiring the DNA-binding domain in the N-terminal of an ancestral periplasmic binding protein (PBP). Phylogenetic analysis of the LacI/GalR family repressors and their PBP homologues revealed that the acquisition of the DNA-binding domain occurred first in the family, and ligand specificity then evolved. The phylogenetic tree also indicates that the acquisition occurred only once before the divergence of the major lineages of eubacteria, and that the LacI/GalR and the PBP families have since undergone extensive gene duplication/loss independently along the evolutionary lineages. Multiple alignments of the repressors and PBPs furthermore revealed that repressors and PBPs with the same ligand specificity have the same or similar residues in their binding sites. This result, together with the phylogenetic relationship, demonstrates that the repressors and the PBPs individually acquired the same ligand specificity by homoplasious replacement, even though their genes are encoded in the same operon.  相似文献   

6.
7.
8.
The short 8–10 amino acid “hinge” sequence in lactose repressor (LacI), present in other LacI/GalR family members, links DNA and inducer‐binding domains. Structural studies of full‐length or truncated LacI‐operator DNA complexes demonstrate insertion of the dimeric helical “hinge” structure at the center of the operator sequence. This association bends the DNA ~40° and aligns flanking semi‐symmetric DNA sites for optimal contact by the N‐terminal helix‐turn‐helix (HtH) sequences within each dimer. In contrast, the hinge region remains unfolded when bound to nonspecific DNA sequences. To determine ability of the hinge helix alone to mediate DNA binding, we examined (i) binding of LacI variants with deletion of residues 1–50 to remove the HtH DNA binding domain or residues 1–58 to remove both HtH and hinge domains and (ii) binding of a synthetic peptide corresponding to the hinge sequence with a Val52Cys substitution that allows reversible dimer formation via a disulfide linkage. Binding affinity for DNA is orders of magnitude lower in the absence of the helix‐turn‐helix domain with its highly positive charge. LacI missing residues 1–50 binds to DNA with ~4‐fold greater affinity for operator than for nonspecific sequences with minimal impact of inducer presence; in contrast, LacI missing residues 1–58 exhibits no detectable affinity for DNA. In oxidized form, the dimeric hinge peptide alone binds to O1 and nonspecific DNA with similarly small difference in affinity; reduction to monomer diminished binding to both O1 and nonspecific targets. These results comport with recent reports regarding LacI hinge interaction with DNA sequences.  相似文献   

9.
10.
The purine repressor is a putative helix-turn-helix DNA-binding protein that regulates several genetic loci important in purine and pyrimidine metabolism in Escherichia coli. The protein is composed of two domains, an N-terminal DNA-binding domain and a C-terminal core that binds the purine co-repressors, guanine and hypoxanthine. The co-repressor binding domain (residues 53 to 341) has been crystallized from polyethylene glycol 600-MgCl2 solutions. They are of the monoclinic form, space group P2(1), with a = 38.2 A, b = 125.7 A, c = 61.8 A and beta = 100.2 degrees. They diffract to a resolution of at least 2.2 A and contain two monomers per asymmetric unit. The importance of the structural determination of this domain is underscored by the high degree of sequence homology displayed within the effector binding sites among a sub-class of helix-turn-helix proteins, of which LacI and GalR are members. The structure of the PurR co-repressor binding domain will provide a high resolution view of one such domain and could serve as a possible model for future effector site structural determinations. Perhaps more important will be this structure's contribution to the further understanding of how protein-DNA interactions are modulated.  相似文献   

11.
The structures and conformational changes of the periplasmic ribose-binding protein and two repressors, PurR and LacI, were compared. Although the closed, ligand-bound structures of the three proteins are very similar, they differ greatly in the degree and direction in which they open, as well as in the amount of internal rearrangement within the domains during that process. Water molecules and a relatively symmetrical inter-domain connection region assist in the large opening observed for the binding protein, while the design of the repressors appears to preclude such dramatic movements. The dimeric nature of the latter proteins, an important aspect in their binding of pseudo-symmetrical DNA sequences, also appears to be a determinant in the allowed motion. Slight differences in the structure of PurR and LacI explain how they can converge to a similar DNA-binding state in response to different binding states of their small molecule effector.  相似文献   

12.
Site-directed mutagenesis was used to change the PurR binding site in the control region of a glyA-lac gene fusion. Mutations that changed the PurR binding sequence away from the consensus sequence reduced PurR binding, which correlated with reduced purine-mediated repression. Mutations that changed the binding sequence toward the consensus sequence had no significant effect on either PurR binding or purine-mediated repression. Hypoxanthine and guanine, co-repressors for PurR-mediated regulation of the pur regulon, increased binding of PurR to glyA operator DNA.  相似文献   

13.
The Escherichia coli purine repressor, PurR, binds to a 16-bp operator sequence and coregulates the genes for de novo synthesis of purine and pyrimidine nucleotides, formation of a one-carbon unit for biosynthesis, and deamination of cytosine. We have characterized the purified repressor. Chemical cross-linking indicates that PurR is dimeric. Each subunit has an N-terminal domain of 52 amino acids for DNA binding and a C-terminal 289-residue domain for corepressor binding. Each domain was isolated after cleavage by trypsin. Sites for dimer formation are present within the corepressor binding domain. The corepressors hypoxanthine and guanine bind cooperatively to distinct sites in each subunit. Competition experiments indicate that binding of one purine abolishes cooperativity and decreases the affinity and the binding of the second corepressor. Binding of each corepressor results in a conformation change in the corepressor binding domain that was detected by intrinsic fluorescence of three tryptophan residues. These experiments characterize PurR as a complex allosteric regulatory protein.  相似文献   

14.
15.
Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19(lacO3/lacOs)), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOS and lacO3 were 5.7 +/- 0.3 x 10(-11) M and 4.1 +/- 0.2 x 10(-11) M respectively, which compare favorably with literature reports of 5 x 10(-10)-1 x 10(-9) M for native lacO1 and 1-1.2 x 10(-10) M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction.  相似文献   

16.
M I Moraitis  H Xu  K S Matthews 《Biochemistry》2001,40(27):8109-8117
Purine repressor (PurR) binding to specific DNA is enhanced by complexing with purines, whereas lactose repressor (LacI) binding is diminished by interaction with inducer sugars despite 30% identity in their protein sequences and highly homologous tertiary structures. Nonetheless, in switching from low- to high-affinity DNA binding, these proteins undergo a similar structural change in which the hinge region connecting the DNA and effector binding domains folds into an alpha-helix and contacts the DNA minor groove. The differences in response to effector for these proteins should be manifest in the polyelectrolyte effect which arises from cations displaced from DNA by interaction with positively charged side chains on a protein and is quantitated by measurement of DNA binding affinity as a function of ion concentration. Consistent with structural data for these proteins, high-affinity operator DNA binding by the PurR-purine complex involved approximately 15 ion pairs, a value significantly greater than that for the corresponding state of LacI (approximately 6 ion pairs). For both proteins, however, conversion to the low-affinity state results in a decrease of approximately 2-fold in the number of cations released per dimeric DNA binding site. Heat capacity changes (DeltaC(p)) that accompany DNA binding, derived from buried apolar surface area, coupled folding, and restriction of motional freedom of polar groups in the interface, also reflect the differences between these homologous repressor proteins. DNA binding of the PurR-guanine complex is accompanied by a DeltaC(p) (-2.8 kcal mol(-1) K(-1)) more negative than that observed previously for LacI (-0.9 to -1.5 kcal mol(-1) K(-1)), suggesting that more extensive protein folding and/or enhanced structural rigidity may occur upon DNA binding for PurR compared to DNA binding for LacI. The differences between these proteins illustrate plasticity of function despite high-level sequence and structural homology and undermine efforts to predict protein behavior on the basis of such similarities.  相似文献   

17.
18.
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.  相似文献   

19.
The gamma-butyrolactone-type autoregulator/receptor systems in the Gram-positive bacterial genus Streptomyces regulate morphological differentiation or antibiotic production, or both. The autoregulator receptors act as DNA-binding proteins, and on binding their cognate ligands (gamma-butyrolactones) they are released from the DNA, thus serving as repressors. The crystal structure of CprB in Streptomyces coelicolor A3(2), a homologue of the A-factor-receptor protein, ArpA, in Streptomyces griseus, was determined. The overall structure of CprB shows that the gamma-butyrolactone receptors belong to the TetR family. CprB is composed of two domains, a DNA-binding domain and a regulatory domain. The regulatory domain contains a hydrophobic cavity, which probably serves as a ligand-binding pocket. On the basis of the crystal structure of CprB and on the analogy of the characteristics of ligand-TetR binding, the binding of gamma-butyrolactones to the regulatory domain of the receptors is supposed to induce the relocation of the DNA-binding domain through conformational changes of residues located between the ligand-binding site and the DNA-binding domain, which would result in the dissociation of the receptors from their target DNA.  相似文献   

20.
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号