Structural and Thermodynamic Dissection of Linear Motif Recognition by the E. coli Sliding Clamp

Abstract

Protein-protein interactions based on linear motif (LM) recognition play roles in many cell regulatory processes. The E. coli sliding clamp is a protein mediator of replisome formation, which uses a common surface pocket composed of two subsites (I and II) to interact with LMs in multiple binding partners. A structural and thermodynamic dissection of sliding clamp-LM recognition has been performed, providing support for a sequential binding model. According to the model, a hydrophobic C-terminal LM dipeptide submotif acts as an anchor to establish initial contacts within subsite I, and this is followed by formation of a stabilizing hydrogen-bonding network between the flanking LM residues and subsite II. Differential solvation/desolvation during positioning of the submotifs is proposed as a driver for the sequential binding. Our model provides general insights into linear motif recognition and should guide the design of small-molecule inhibitors of the E. coli sliding clamp, an emerging antibacterial target.

Differential Modes of Peptide Binding onto Replicative Sliding Clamps from Various Bacterial Origins

“…Interestingly, the orientation of EcM 362 differs between the apo and peptide bound structures of Ec SC 10 and has been shown to be in dynamic equilibrium between these two conformations. 11 These essential interactions are maintained in all SC/ peptides complexes studied, whether the structural information is available experimentally or by docking of the native peptide on the apo SC structures (see SI.12A,B,E). However, the changes in sequence between the different SC may affect the detailed energetic balance between the different interaction sites.…”

“…10 Recently, it was suggested that peptide binding onto Ec SC could occur via a two steps process, with an initial interaction in SS1, followed by the binding in SS2. 11 Because SC is a central element for the physiological functions of enzymes involved in DNA metabolism 4 and their trafficking, 12,13 the binding pocket has been considered as a target for the development of new antibacterial drugs. 5,9,14 Several studies have explored this potentiality either by screening a chemical bank 14 or by a structured-based design approach, opening the way for structure−activity studies.…”

Crystal structures and biochemical characterization of DNA sliding clamps from three Gram-negative bacterial pathogens

“…Linear motif interactions with  Eco have been extensively characterized (Wijffels et al, 2004;Georgescu et al, 2008b;Wolff et al, 2011;Yin et al, 2013), and interactions between linear motif peptides and  from Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus, and P. aeruginosa, while less studied, have been reported (Gui et al, 2011;Wolff et al, 2014). In the present study, binding of a fluorescently labeled linear motif-consensus peptide (5FAM-Q1L2D3L4F5-OH; "P1") to the bacterial  clamps studied by fluorescence polarization (FP).…”

“…FP experiments were carried out as described previously (Yin et al, 2013(Yin et al, , 2014(Yin et al, , 2015. The fluorescent peptides, at 10 nM concentration, and proteins were in 10 mM HEPES buffer, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, 0.07% (v/v) nonidet-P40 and 5% (v/v) DMSO.…”

“…Subsite I binds linear motif residues Lx2F/L and subsite II binds the QL dipeptide, with residue x1 linking these groups between the two subsites. We have proposed an 'anchor-based' model of linear motif binding to the Escherichia coli  in which the C-terminal (Lx2F/L) motif first binds to subsite I (the "anchor site"), followed by binding of the QL motif into subsite II (Yin et al, 2013). Non-peptidic inhibitors, including a thioxothiazolinine derivative (Georgescu et al, 2008b) and a biphenyloxime ether (Wijffels et al, 2011), have been shown to occupy subsite I only, whereas the natural cyclic peptide antibiotic griselimycin is able to occupy both subsites in the Mycobacterium smegmatis  (Kling et al, 2015).…”