The HNH Family A Tree of Three Branches 21 The HNH Consensus Sequence

The -34 amino acid HNH motif gained its name from the invariant histidine and asparagine residues that are characteristic of the motif. Since first described, many new sequences have been added to the group allowing a more

Fig. 1. Consensus sequence and secondary structure of the HNH motif. 1 Consensus sequence for the HNH motif. Residues in bold are those that denote the motif itself, underlined residues indicate positions where hydrophobic residues stabilize the fold in the context of the recipient protein scaffold (see Fig. 3). Residues are numbered according to the motif in the colicin E9 DNase domain. The secondary structure of the motif is indicated above the consensus sequence. 2 Structure of the histidine-rich HNH motif from the colicin E9 DNase bound to a single Zn2+ ion (1FSJ). The HNH motif is denoted by Hisl03 (general base), Asnll8 (secondary structure stabilization) and Hisl27 (metal ion ligand), respectively. GlulOO and Hisl31 form the semi-conserved Glu and His within the EXjHH-HX3H motif of Gorbalenya (1994). GlulOO is at the center of a hydrogen-bond network involving both Hisl27 and Arg96, which are not strictly part of the HNH motif but used in the E9 DNase for binding and distorting the minor groove of DNA. Hisl31 is only a metal ion ligand in the context of the Zn2+-bound structure of the E9 DNase. For HNN enzymes, the metal ion ligands Hisl02 and Hisl27 are replaced by aspartate or asparagine and asparagine, respectively

Fig. 1. Consensus sequence and secondary structure of the HNH motif. 1 Consensus sequence for the HNH motif. Residues in bold are those that denote the motif itself, underlined residues indicate positions where hydrophobic residues stabilize the fold in the context of the recipient protein scaffold (see Fig. 3). Residues are numbered according to the motif in the colicin E9 DNase domain. The secondary structure of the motif is indicated above the consensus sequence. 2 Structure of the histidine-rich HNH motif from the colicin E9 DNase bound to a single Zn2+ ion (1FSJ). The HNH motif is denoted by Hisl03 (general base), Asnll8 (secondary structure stabilization) and Hisl27 (metal ion ligand), respectively. GlulOO and Hisl31 form the semi-conserved Glu and His within the EXjHH-HX3H motif of Gorbalenya (1994). GlulOO is at the center of a hydrogen-bond network involving both Hisl27 and Arg96, which are not strictly part of the HNH motif but used in the E9 DNase for binding and distorting the minor groove of DNA. Hisl31 is only a metal ion ligand in the context of the Zn2+-bound structure of the E9 DNase. For HNN enzymes, the metal ion ligands Hisl02 and Hisl27 are replaced by aspartate or asparagine and asparagine, respectively refined consensus sequence to be established (Fig. 1.1). Described from the N-terminus, the HNH motif comprises five elements: (1) an absolutely conserved histidine (HNH), usually part of a His-His or Asp-His dyad, with this dyad flanked by hydrophobic residues; (2) a Gly/Pro loop of varying length; (3) an invariant asparagine (HNH) followed by a leucine residue; (4) a region of variable length (X5 7) and irregular structure that contains one or more hydrophobic residues involved in stabilizing the motif; and (5) a histidine (HNH). Alternative residues at key positions are also shown in Fig. 1, notable amongst these are a Glu/Gln/Asn residue immediately N-terminal to the hydrophobic residue flanking the His-His dyad, and an additional histidine (or glutamine) 3-4 residues C-terminal to the HNH residue. It was in this form that the HNH motif (EXjHH-HX^) was described by Gorbalenya (1994). Mutagenesis experiments have confirmed the importance of many of these residues for catalytic activity toward DNA, including the His-His dyad, the HNH residue, and the glutamate and C-terminal histidine residues (Walker et al. 2002).

Most of the available crystal structures of HNH-containing enzymes are of DNA-degrading bacterial toxins called colicins. These are plasmid-encod-ed toxins produced by Escherichia coli during times of stress to kill competing strains (James et al. 2002). Crystal structures of the endonuclease domains of colicin E7 (Ko et al. 1999) and E9 (Kleanthous et al. 1999) show that the HNH motif is composed of two P-strands and an a-helix with a metal ion sandwiched between the structural elements (Fig. 1.2), an arrangement resembling a Zn2+ finger (Grishin 2001). A single transition metal ion can bind within the histidine-rich HNH motif adopting tetrahedral geometry (Kleanthous et al. 1999; Ko et al. 1999), although the physiological relevance of this has proven controversial (see Sects. 3 and 4). The transition metal ligating residues project from the secondary structural elements themselves (in contrast to conventional Zn2+ fingers where the metal ion is coordinated by residues presented on loops) and comprise the first histidine of the His-His dyad, the HNH histidine, and the C-terminal histidine residue (Fig. 1). The asparagine of the motif (HNH) serves a structural role forming a backbone hydrogen bond across the motif (Kleanthous et al. 1999; Ko et al. 1999).

2.2 The Cysteine-Containing HNH Motif (cysHNH)

A large number of HNH enzymes, including the nrdB intron-encoded endonuclease I-TevIII and 5-methylcytosine-dependent restriction endonuclease McrA (Eddy and Gold 1991; Shub et al. 1994), have additional consensus sequences that take the form of two CX2 4C motifs, one 8-12 residues N-ter-minal to the His-His dyad and the other adjacent to the HNH residue (not shown). Modelling studies suggest that these cysteines point away from the active site and coordinate structural Zn2+ ions (Bujnicki et al. 2000), a feature that is shared with His-Cys enzymes such as I-Ppol (see Galburt and Jurica, this Vol.). We suggest such motifs are denoted as cysHNH motifs to distinguish them from the general HNH group of enzymes.

2.3 The HNN Variant

A further variation of the HNH motif exists that is modified in two respects: (1) the initial His-His dyad is replaced by an Asp-His or Asn-His pair; and (2) the HNH residue is replaced by an asparagine to form an HNN motif. These substitutions are highly relevant since the two changed residues ligate the active site metal ion (Maté and Kleanthous 2004; Fig. 1.2). The HNN variant can also be found in combination with two stabilizing CX2 4C couplets (cy-sHNN).

2.4 The HNH Motif Is Part of the Wider ppa-Me Super family of Endonucleases

Conservation of the HNH motif fold extends across a wider range of endonucleases, alternatively termed the ppa-Me or His-Me finger endonucleases (Kühlmann et al. 1999; Aravind et al. 2000). This structural homology was first identified by Kühlmann et al. (1999), who reported that the active sites of the His-Cys homing endonuclease I-Ppol (Fig. 2) and the non-specific nuclease from Serratia superimposed with that of the E9 DNase with a rmsd for main chain atoms of 1.2 and 1.5 Á, respectively. The central Asn-Leu pair of the HNH motif is also present in Serratia but replaced by a His-Leu pair in I-Ppol, in each case the polar residue stabilizing the motif fold. I-Ppol is a cy-sHNN variant while Serratia nuclease is an HNN motif enzyme. Many PPa-Me motif enzymes belong to the HNN group, including the Holliday junction resolving enzyme T4 endo VII (Aravind et al. 2000).

Other notable HNH/ppa-Me endonucleases are the apoptotic enzyme CAD, the periplasmic nuclease Vvn from Vibrio vulnificus and the homing endonuclease I-Hmul (Li et al. 2003; Shen et al. 2004; Woo et al. 2004). Figure 2 shows a comparison of the structures of CAD and Vvn alongside those of I-Ppol and the E9 DNase. It illustrates the similarity of the ppa-Me motif in enzymes that are otherwise structurally unrelated. The individual motifs are accommodated on the different enzyme scaffolds through the burial of 46 conserved (or conservatively substituted) hydrophobic amino acids. These

Fig. 2. The HNH/ppa-Me motif in different endonucleases. The figure shows the crystal structures of avarietyof enzymes containingthe motif (black). 1 Colicin E9 DNase (1FSJ); 2 CAD (1V0D); 3 Vvn (10U0); 4 I-Ppol (1CYQ). The figure also highlights how a large section of non-conserved sequence connecting the p-strands of the motif adopts a variety of folds, forming an irregular structure in the E9 DNase and I-Ppol but folding into a-helices in CAD and Vvn

Fig. 2. The HNH/ppa-Me motif in different endonucleases. The figure shows the crystal structures of avarietyof enzymes containingthe motif (black). 1 Colicin E9 DNase (1FSJ); 2 CAD (1V0D); 3 Vvn (10U0); 4 I-Ppol (1CYQ). The figure also highlights how a large section of non-conserved sequence connecting the p-strands of the motif adopts a variety of folds, forming an irregular structure in the E9 DNase and I-Ppol but folding into a-helices in CAD and Vvn are shown in Fig. 3.1 for the colicin E9 DNase, with a corresponding structural superposition for the E9 DNase and CAD presented in Fig. 3.2. The hydrophobic residues dock into pockets in the recipient protein, essentially bolting the motif to the protein. The ability to dock the motif onto differing scaffolds explains its wide distribution in evolutionarily unrelated enzymes.

Fig. 3. The HNH/ppa-Me motif is bolted onto different protein scaffolds by hydrophobic residues that dock into pockets in the host protein. 1 The HNH motif of the colicin E9 DNase is shown in ribbon while the remainder of the molecule is shown as a molecular surface. The motif is accommodated through the insertion of (mostly conserved) hydrophobic residues into corresponding pockets in the E9 DNase. 2 Structural overlay of the HNH/ ppa-Me motifs of the colicin E9 DNase (black; 1EMV) and CAD (gray, 1V0D) showing the positions of the key hydrophobic residues (numbered according to the E9 DNase) that enable the motif to be bolted onto different protein scaffolds

Fig. 3. The HNH/ppa-Me motif is bolted onto different protein scaffolds by hydrophobic residues that dock into pockets in the host protein. 1 The HNH motif of the colicin E9 DNase is shown in ribbon while the remainder of the molecule is shown as a molecular surface. The motif is accommodated through the insertion of (mostly conserved) hydrophobic residues into corresponding pockets in the E9 DNase. 2 Structural overlay of the HNH/ ppa-Me motifs of the colicin E9 DNase (black; 1EMV) and CAD (gray, 1V0D) showing the positions of the key hydrophobic residues (numbered according to the E9 DNase) that enable the motif to be bolted onto different protein scaffolds

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