Structural basis for the nucleic acid binding cooperativity of bacteriophage T4 gene 32 protein: the (Lys/Arg)3(Ser/Thr)2 (LAST) motif.

Abstract

To identify the functional residues of the N-terminal B region of bacteriophage T4 gene 32 protein involved in its cooperative binding to single-stranded nucleic acids, a process dependent on homotypic protein-protein interaction, we have studied the interaction of the protein with synthetic peptides containing different portions of this domain. Gel-permeation chromatography showed that a 6-acryloyl-2-dimethylaminonaphthalene (acrylodan)-labeled fluorescent peptide corresponding to the first 17 residues of gene 32 protein formed a complex with whole protein. The fluorescence was blue-shifted 14 nm upon interaction with intact protein, and somewhat less so (7-11 nm) with cleavage products of the protein lacking B domains. The intrinsic tryptophan fluorescence of whole and truncated protein was quenched by this peptide and by the nonderivatized peptide. The peptide bound tightly to truncated protein at both 0.015 and 0.44 M Na+, with a stoichiometry of 1:1. Similar tryptophan quenching or acrylodan blue shifts were obtained with peptides corresponding to residues 1-9 and 3-8, but not residues 1-4, 5-9, or 5-17, indicating that the essential amino acids are contained within positions 3-8, Lys-Arg-Lys-Ser-Thr-Ala. Several other DNA binding proteins contain a LAST motif with documented involvement of these residues in nucleic acid interaction. The amino acid and coding sequence of residues 110-114, a region proposed to be involved in nucleic acid binding, is virtually identical to that of residues 3-7. Based on these observations, we have formulated a model for the cooperative interactions of gene 32 protein with single-stranded nucleic acids.

Snapshot of the Genome of the Pseudo-T-Even Bacteriophage RB49

“…The central domain of the T4 gp32 contains both a zinc-finger motif and a fairly regularly spaced series of 6 tyrosine residues, features that are believed to be important to its nucleic acid binding activity (23,24). A second LAST motif is also present here and would be able to interact with either the acidic carboxyl terminal part of the same monomer when the protein is not bound to DNA, or with the nucleic acid backbone when the protein is bound to DNA (15).…”

“…The T4 protein is organized in a three-domain structure. The amino terminal domain of the protein is composed of basic residues and contains a LAST motif (Lys/Arg/Lys/Ser/Thr) (15). This element is believed to be involved in the protein's cooperative binding to DNA (25,48,69,73,79).…”

Mechanical Measurement of Single-molecule Binding Rates: Kinetics of DNA Helix-destabilization by T4 Gene 32 Protein

“…It has been suggested that an acidic arm of the core domain adjacent to the C-domain (within residues 200-239) might occupy the trough between the two lobes of gp32, and would therefore have to be displaced in order for ssDNA to bind. 19,43 This trough contains a patch of cationic residues Lys-Arg-Lys-Thr-Ser (the LAST motif) 43,46,47 at positions 110-114 of the core domain that was shown to bind directly to ssDNA. 17,43,46,48 In the full-length protein, displacement would involve the adjacent acidic C-domain (residues 254-301), which might find additional basic regions of the core to associate with, and thus further slow the binding process.…”

Salt Dependent Binding of T4 Gene 32 Protein to Single and Double-stranded DNA: Single Molecule Force Spectroscopy Measurements

“…In the absence of binding nucleic acid, the CTD could occupy the cationic trough between the two lobes of gp32 protein, where residues 110-114, Lys-Arg-Lys-Thr-Ser, were shown to bind directly to ssDNA. 3,21,44,45 The CTD would therefore have to move away in order for the DNA to bind. This model is also supported by the results of the study of the effect of the CTD on the proteolytic accessibility of the protein in the DNA-bound and unbound forms.…”