Details of the Nucleic Acid Binding Site of T4 Gene 32 Protein Revealed by Proteolysis and DNA T m Depression Methods Min Wu, Elizabeth K. Flynn and Richard L. Karpel* Department of Chemistry and Biochemistry, University of Maryland, Baltimore County Baltimore, MD 21250, USA The af®nities and location of oligonucleotides bound to intact and trun- cated bacteriophage T4 gene 32 protein have been elucidated by two independent and sensitive methods. The nucleic acid binding site is located within the core domain of 32 protein, residues 22-253. Oligonu- cleotides protect the core domain against proteolysis catalyzed by mam- malian endoproteinase Arg-C. Of the three cleavage sites, Arg111, within the internal ``LAST'' ((Lys/Arg) 3 (Ser/Thr) 2 ) motif, is selectively protected. We have previously suggested that these LAST residues, Lys-Arg-Lys- Thr-Ser, residues 110-114, are involved in nucleic acid binding, and our results are also consistent with crystallographic studies. The inhibitory effects of oligonucleotides on the kinetics of core domain proteolysis were used to quantify binding af®nities. In addition, af®nities of oligonu- cleotides for both core domain and intact protein were obtained from their effect on the T m -depressing activities of these proteins. For both core and intact protein, the degree of af®nity increases with oligonucleo- tide length. The presence of a 5 0 terminal phosphate increases the af®nity two- to fourfold. Placement of methylphosphonodiester (uncharged) lin- kages at alternating linkages vastly lowers binding af®nity for the intact protein and core domain. We conclude that at least two and likely three adjacent phosphodiester linkages are a minimal requirement for binding, further de®ning the electrostatic component of the interaction. The length-dependence of binding af®nity suggests that additional interactions, both ionic and non-ionic, likely occur with longer oligonu- cleotides. # 1999 Academic Press Keywords: gene 32 protein; DNA; oligonucleotide; proteolysis; T m *Corresponding author Introduction Bacteriophage T4 gene 32 protein is a single- strand-speci®c nucleic acid binding protein, and is a model for this class of proteins (for a review, see Karpel, 1990). Gene 32 protein binds cooperatively to single-stranded nucleic acids, a property depen- dent on homotypic protein-protein interactions. The results of a variety of experiments strongly indicate that the nucleic acid binding surface is contained entirely within residues 22-253 of the protein, which structurally and functionally de®ne the ``core'' domain, sometimes referred to as * III (Karpel, 1990). The core domain binds nucleic acids non-cooperatively, and the af®nities associ- ated with these interactions are about 1000-fold lower than the corresponding values for full-length protein; the differences can be attributed largely to the lack of binding cooperativity (Kowalcykowski et al., 1981). Cooperativity is clearly dependent on the presence of the N-terminal domain, since removal of the ®rst 21 residues of the protein (with trypsin), or as few as nine residues (with V8 pro- E-mail address of the corresponding author: karpel@umbc.edu Abbreviations used: 32 protein, bacteriophage T4 gene 32 protein; * III, central or ``core'' domain of 32 protein, containing the nucleic acid binding site; poly[d(A-T)], double-stranded polydeoxyribonucleotide with alternating A-T sequence; [poly[d(A-T)]] p , concentration of (in this case) poly[d(A-T)] in nucleotide residue mol/ l; LAST motif, (Lys/Arg) 3 (Ser/Thr) 2 sequence found twice in 32 protein; d(TrTp) 4 TrT, decadeoxythymidylate analog containing alternating phosphonodiester linkages; PVDF, polyvinylidene ¯uoride; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid. Article No. jmbi.1999.2541 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 286, 1107±1121 0022-2836/99/091107±15 $30.00/0 # 1999 Academic Press