PDBsum entry 1c1k

DNA binding protein

PDB id

1c1k

Contents

			Protein chain

					217 a.a. *

			Metals

					_CL ×12


					_IR ×2

			Waters ×298

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Bacteriophage t4 gene 59 helicase assembly protein binds replication fork DNA. The 1.45 a resolution crystal structure reveals a novel alpha-Helical two-Domain fold.

Authors

T.C.Mueser, C.E.Jones, N.G.Nossal, C.C.Hyde.

Ref.

J Mol Biol, 2000, 296, 597-612. [DOI no: 10.1006/jmbi.1999.3438]

PubMed id

10669611

Abstract

The bacteriophage T4 gene 59 helicase assembly protein is required for recombination-dependent DNA replication, which is the predominant mode of DNA replication in the late stage of T4 infection. T4 gene 59 helicase assembly protein accelerates the loading of the T4 gene 41 helicase during DNA synthesis by the T4 replication system in vitro. T4 gene 59 helicase assembly protein binds to both T4 gene 41 helicase and T4 gene 32 single-stranded DNA binding protein, and to single and double-stranded DNA. We show here that T4 gene 59 helicase assembly protein binds most tightly to fork DNA substrates, with either single or almost entirely double-stranded arms. Our studies suggest that the helicase assembly protein is responsible for loading T4 gene 41 helicase specifically at replication forks, and that its binding sites for each arm must hold more than six, but not more than 12 nucleotides. The 1.45 A resolution crystal structure of the full-length 217-residue monomeric T4 gene 59 helicase assembly protein reveals a novel alpha-helical bundle fold with two domains of similar size. Surface residues are predominantly basic (pI 9.37) with clusters of acidic residues but exposed hydrophobic residues suggest sites for potential contact with DNA and with other protein molecules. The N-terminal domain has structural similarity to the double-stranded DNA binding domain of rat HMG1A. We propose a speculative model of how the T4 gene 59 helicase assembly protein might bind to fork DNA based on the similarity to HMG1, the location of the basic and hydrophobic regions, and the site size of the fork arms needed for tight fork DNA binding. The fork-binding model suggests putative binding sites for the T4 gene 32 single-stranded DNA binding protein and for the hexameric T4 gene 41 helicase assembly.



	Figure 3. Figure 3. T4 gene 59 protein N-domain. (a) The 1.45 Å resolution 2F[o] -F[c] electron density map (contoured at 1s) in the N-domain region showing Trp41 in loop H1-H2 packed against Pro7 in loop S1-H1. (b) "Top" view trace of the N-domain with side-chains of a basic cluster (Lys52, Arg53, Arg54, Lys56, Lys61, Lys65, Lys67, and Lys69) and an acidic cluster (Asp55, Asp84, Asp89, Asp92, and Asp94). Asp94, an a[L] conformer, is surrounded by exposed hydrophobic residues (Leu96, Val97 and Phe98). Asp84 is adjacent to Ile87 and Trp86 at the domain interface. A polar residue, Glu159, buried in the interface is hydrogen bonded to the phenol oxygen atoms of Tyr102 and Tyr193. (c) "Bottom" view of the N-domain trace with side-chains of Asn40 and Trp41 near exposed hydrophobic regions (residues Leu4, Met6, Pro7, Ile14, Leu22, Met25, Leu68, Leu73, Ile103, Leu106, and Ile109) . The side-chain of Phe111, located on the first turn of helix H7 is pointing directly into the solvent space, unaffected by crystal lattice contacts. Ile109 packs against Ile2 of strand S1 anchoring the N-terminal b-strand.		Figure 4. Figure 4. T4 gene 59 protein C-domain. (a) A 1.45 Å resolution 2F[o] -F[c] electron density map (contoured at 1s) of the loop H8-H9 region where Pro140 is the first residue (or the "i position") of an extended b-hairpin. The quality of the map ensures proper assignment of the a[L] conformation of Gln143 (the fourth residue of the b-hairpin, b i+3) with Val142 (b i+2) directed towards a solvent-accessible hydrophobic surface. (b) "Top" view of the C-domain trace shows side-chains of several a[L] residues (Glu128 in loop H7-H8, Gln143 in H8-H9, Asn154 in H9-H10) and a cluster of exposed hydrophobic residues (Phe123, Val127, Val129, Phe148, Leu150, Ile155, Leu181, Val182, and Ile188) form the "top" surface of the C-domain. (c) "Bottom" view of the C-domain trace shows the side-chains of the a[L] conformation residue Asn169 and solvent-exposed residues (Phe132, Phe136, Phe167, Phe208 and Ile209) clustered in a hydrophobic patch forming the "bottom" surface of the C-domain. An acidic patch (Asp165, Asp172, Asp175, Glu176, and Asp179) is adjacent to a large cluster of basic residues located within the central groove (Arg5, Lys191, Arg194, and Lys195).

PROCHECK

	Headers