A context-dependent ClpX recognition determinant located at the C terminus of phage Mu repressor

The bacteriophage Mu immunity repressor is a conformationally sensitive sensor that can be interconverted between forms resistant to and sensitive to degradation by ClpXP protease. Protease-sensitive repressor molecules with an altered C-terminal sequence promote rapid degradation of the wild-type repressor by inducing its C-terminal end to become exposed. Here we determined that the last 5 C-terminal residues (CTD5) of the wild-type repressor contain the motif required for recognition by the ClpX molecular chaperone, a motif that is strongly dependent upon the context in which it is presented. Although attachment of the 11-residue ssrA degradation tag to the C terminus of green fluorescent protein (GFP) promoted its rapid degradation by ClpXP, attachment of 5-27 C-terminal residues of the repressor failed to promote degradation. Disordered peptides derived from 41 and 35 C-terminal residues of CcdA (CcdA41) and thioredoxin (TrxA35), respectively, activated CTD5 when placed as linkers between GFP and repressor C-terminal sequences. However, when the entire thioredoxin sequence was included as a linker to promote an ordered configuration of the TrxA35 peptide, the resulting substrate was not degraded. In addition, a hybrid tag, in which CTD5 replaced the 3-residue recognition motif of the ssrA tag, was inactive when attached directly to GFP but active when attached through the CcdA41 peptide. Thus, CTD5 is sufficient to act as a recognition motif but has requirements for its presentation not shared by the ssrA tag. We suggest that activation of CTD5 may require presentation on a disordered or flexible domain that confers ligand flexibility.     

Activation of a dormant ClpX recognition motif of bacteriophage Mu repressor by inducing high local flexibility

The C-terminal domain (CTD) of bacteriophage Mu immunity repressor (Rep) regulates DNA binding by the N-terminal domain and degradation by ClpXP protease. Five residues at the Rep C terminus (CTD5) can serve as a ClpX recognition motif, but it is dormant unless activated, a state that can be induced by the presence of dominant-negative mutant repressors (Vir). Conversion of Rep to ClpXP-sensitive form was associated with not only increased exposure of CTD5 to solvent but also increased CTD motion or flexibility as measured by fluorescence anisotropy. CTD mutations (V183S, K193S, and V196S) promoting ClpXP resistance without destroying the recognition motif prevented increased CTD motion induced by Vir. Suppression of ClpXP protease resistance conferred by the V196S mutation also correlated with restoration of CTD motion. The temperature-sensitive R47Q mutation present in cis within the DNA-binding domain restored ClpXP protease sensitivity to the V196S mutant, and anisotropy analysis indicated that R47Q allows the V196S CTD to gain increased flexibility when Vir was present. The results indicate that the CTD functions to turn the recognition motif on and off, most likely by modulating flexibility of the domain that harbors the ClpX recognition motif, suggesting a general mechanism by which proteins can regulate their own degradation.     

Modulation of phage Mu repressor DNA binding and degradation by distinct determinants in its C-terminal domain

Rapid degradation of the bacteriophage Mu immunity repressor can be induced in trans by mutant, protease-hypersensitive repressors (Vir) with an altered C-terminal domain (CTD). Genetic and biochemical analysis established that distinct yet overlapping determinants in the wild-type repressor CTD modulate Vir-induced degradation by Escherichia coli ClpXP protease and DNA binding by the N-terminal DNA-binding domain (DBD). Although deletions of the repressor C-terminus resulted in both resistance to ClpXP protease and suppression of a temperature-sensitive DBD mutation (cts62), some cysteine-replacement mutations in the CTD elicited only one of the two phenotypes. Some CTD mutations prevented degradation induced by Vir and resulted in the loss of intrinsic ClpXP protease sensitivity, characteristic of wild-type repressor, and at least two mutant repressors protected Vir from proteolysis. One protease-resistant mutant became susceptible to Vir-induced degradation when it also contained the cts62 mutation, which weakens DNA binding but apparently facilitates conversion to a protease-sensitive conformation. Conversely, this CTD mutation was able to suppress temperature sensitivity of DNA binding by the cts62 repressor. The results suggest that determinants in the CTD not only provide a cryptic ClpX recognition motif but also direct CTD movement that exposes the motif and modulates DNA binding.     

DNA recognition by metal-peptide complexes containing the recognition helix of the phage 434 repressor

 Short oligopeptides (14 residues) derived from the DNA recognition helix of the phage 434 repressor (434R) have been tethered onto the metallointercalating [Rh(phi)₂(phen′)]³⁺, and the DNA recognition characteristics of the resultant metal-peptide complexes have been examined. DNA sequence selectivities for the family of metal-peptide complexes, determined in photoactivated DNA cleavage experiments, reproduce features of operator recognition by the native 434R. Binding to the DNA duplex depends both on the appended peptide and upon the metallointercalating unit, as determined through variations in the peptide sequence and in the diastereomeric configuration of the metal-peptide. The complexes preferentially target 5′-ACAA-3′ operator sites and single-base variants, bind at 50 nM concentrations, and, as determined by chemical footprinting, protect 7–10 bp of DNA around the target sites. Comparative cleavage studies on synthetic oligonucleotides containing variations in operator sequence, furthermore, reveal a hierarchy in sequence preference which resembles the native protein, but highest affinity for the metal-peptides, unlike 434R, is found for 5′-ACGA-3′. These studies illustrate a new route to the rational design of small, artificial repressors through the construction of metal-peptide complexes. Received: 18 June 1997 / Accepted: 11 September 1997     

Construction of plasmids that produce phage P22 repressor

In a series of plasmid constructions, the c2 (repressor) gene of phage P22 was cloned in a multicopy plasmid and expressed at increasing level. The final result of these constructions is a plasmid that maintains a level of approx. 200 times as much repressor as is found in a lysogen. A series of increasingly virulent phage mutants was isolated by plating sequentially on host cells with increasing levels of repressor. The methods used in the constructions should be applicable to obtaining elevated expression of cloned genes in other systems.     

Inhibition of bacteriophage Mu transposition by Mu repressor and Fis

In this paper we show that the Escherichia coli protein Fis has a regulatory function in Mu transposition in the presence of Mu repressor. Fis can lower the transposition frequency of a mini-Mu 3–80-fold, but only if the Mu repressor is expressed simultaneously. In this novel type of regulation of transposition by the concerted action of Fis and repressor, the IAS, the internal activating sequence, is also involved as deletion of this site leads to the loss of the Fis effect. As the IAS contains strong repressor binding sites these are probably the target for the repressor in the observed negative regulation by Fis and repressor. However, the role of Fis and repressor is not only to inactivate the IAS, since a 4bp insertion in the IAS, which changes the spacing of the repressor-binding site, abolishes the enhancing function of the IAS but leaves the repressor-Fis effect intact. A likely target for Fis in this regulation is a strong Fis-binding site, which is located adjacent to the L2 transposase-binding site. However, when this Fis-binding sequence was substituted by a random sequence and Fis no longer showed specific binding to this site, the Fis effect was still observed. Although it is still possible that Fis can function by binding to this non-specific site in a particular complex, it seems more likely that Fis is directly or indirectly involved in determining the level of the repressor.     

Solution structure of the Mu end DNA-binding ibeta subdomain of phage Mu transposase: modular DNA recognition by two tethered domains.

The phage Mu transposase (MuA) binds to the ends of the Mu genome during the assembly of higher order nucleoprotein complexes. We investigate the structure and function of the MuA end-binding domain (Ibetagamma). The three-dimensional solution structure of the Ibeta subdomain (residues 77-174) has been determined using multidimensional NMR spectroscopy. It comprises five alpha-helices, including a helix-turn-helix (HTH) DNA-binding motif formed by helices 3 and 4, and can be subdivided into two interacting structural elements. The structure has an elongated disc-like appearance from which protrudes the recognition helix of the HTH motif. The topology of helices 2-4 is very similar to that of helices 1-3 of the previously determined solution structure of the MuA Igamma subdomain and to that of the homeodomain family of HTH DNA-binding proteins. We show that each of the two subdomains binds to one half of the 22 bp recognition sequence, Ibeta to the more conserved Mu end distal half (beta subsite) and Igamma to the Mu end proximal half (gamma subsite) of the consensus Mu end-binding site. The complete Ibetagamma domain binds the recognition sequence with a 100- to 1000-fold higher affinity than the two subdomains independently, indicating a cooperative effect. Our results show that the Mu end DNA-binding domain of MuA has a modular organization, with each module acting on a specific part of the 22 bp binding site. Based on the present binding data and the structures of the Ibeta and Igamma subdomains, a model for the interaction of the complete Ibetagamma domain with DNA is proposed.     

Kinetic evidence that allosteric activation of antithrombin by heparin is mediated by two sequential conformational changes

The serpin, antithrombin, requires allosteric activation by a sequence-specific pentasaccharide unit of heparin or heparan sulfate glycosaminoglycans to function as an anticoagulant regulator of blood clotting proteases. Surprisingly, X-ray structures have shown that the pentasaccharide produces similar induced-fit changes in the heparin binding site of native and latent antithrombin despite large differences in the heparin affinity and global conformation of these two forms. Here we present kinetic evidence for similar induced-fit mechanisms of pentasaccharide binding to native and latent antithrombins and kinetic simulations which together support a three-step mechanism of allosteric activation of native antithrombin involving two successive conformational changes. Equilibrium binding studies of pentasaccharide interactions with native and latent antithrombins and the salt dependence of these interactions suggest that each conformational change is associated with distinct spectroscopic changes and is driven by a progressively better fit of the pentasaccharide in the binding site. The observation that variant antithrombins that cannot undergo the second conformational change bind the pentasaccharide like latent antithrombin and are partially activated suggests that both conformational changes contribute to allosteric activation, in agreement with a recently proposed model of allosteric activation.     

Frameshift mutations in the bacteriophage Mu repressor gene can confer a trans-dominant virulent phenotype to the phage.

Virulent mutations in the bacteriophage Mu repressor gene were isolated and characterized. Recombination and DNA sequence analysis have revealed that virulence is due to unusual frameshift mutations which change several C-terminal amino acids. The vir mutations are in the same repressor region as the sts amber mutations which, by eliminating several C-terminal amino acids, suppress thermosensitivity of repressor binding to the operators by its N-terminal domain (J. L. Vogel, N. P. Higgins, L. Desmet, V. Geuskens, and A. Toussaint, unpublished data). Vir repressors bind Mu operators very poorly. Thus the Mu repressor C terminus, either by itself or in conjunction with other phage or host proteins, tunes the DNA-binding properties at the repressor N terminus.     

DNA sequence of the control region of phage D108: the N-terminal amino acid sequences of repressor and transposase are similar both in phage D108 and in its relative, phage Mu.

We have determined the DNA sequence of the control region of phage D108 up to position 1419 at the left end of the phage genome. Open reading frames for the repressor gene, ner gene, and the 5' part of the A gene (which codes for transposase) are found in the sequence. The genetic organization of this region of phage D108 is quite similar to that of phage Mu in spite of considerable divergence, both in the nucleotide sequence and in the amino acid sequences of the regulatory proteins of the two phages. The N-terminal amino acid sequences of the transposases of the two phages also share only limited homology. On the other hand, a significant amino acid sequence homology was found within each phage between the N-terminal parts of the repressor and transposase. We propose that the N-terminal domains of the repressor and transposase of each phage interact functionally in the process of making the decision between the lytic and the lysogenic mode of growth.     

Structural basis of SspB-tail recognition by the zinc binding domain of ClpX

The degradation of ssrA(AANDENYALAA)-tagged proteins in the bacterial cytosol is carried out by the ClpXP protease and is markedly stimulated by the SspB adaptor protein. It has previously been reported that the amino-terminal zinc-binding domain of ClpX (ZBD) is involved in complex formation with the SspB-tail (XB: ClpX-binding motif). In an effort to better understand the recognition of SspB by ClpX and the mechanism of delivery of ssrA-tagged substrates to ClpXP, we have determined the structures of ZBD alone at 1.5, 2.0, and 2.5 A resolution in each different crystal form and also in complex with XB peptide at 1.6 A resolution. The XB peptide forms an antiparallel beta-sheet with two beta-strands of ZBD, and the structure shows a 1:1 stoichiometric complex between ZBD and XB, suggesting that there are two independent SspB-tail-binding sites in ZBD. The high-resolution ZBD:XB complex structure, in combination with biochemical analyses, can account for key determinants in the recognition of the SspB-tail by ClpX and sheds light on the mechanism of delivery of target proteins to the prokaryotic degradation machine.