Enzymology of HNHpaMe Motif Endonucleases

3.1 The HNH/pp«-Me Motif Is Functionally Adaptable

Enzymes that contain the HNH/ppa-Me motif and no other DNA recognition domains, as is the case for the bacterial colicins (Fig. 4.1), CAD and Serratia nuclease, show little cleavage specificity. This is consistent with the biological roles for these proteins whose function it is to degrade intracellular DNA (colicins, CAD), or, as in the case of Serratia nuclease, catabolize extracellular nucleic acids for nutrient uptake (Friedhoff et al. 1999; Widlak et al. 2000; James et al. 2002). In some cases (Serratia, colicins), this low specificity extends to cleaving ssDNA and RNA (Friedhoff et al. 1999; Pommer et al. 2001). DNA recognition specificity is essential for the homing function of intron-encoded endonucleases, with this specificity emanating from DNA recognition domains. For example, in addition to its HNH motif, I-Hmul contains three such domains (Fig. 4.2): (1) an N-terminal NUMOD4 (Nuclease-associated MODule, type 4) domain comprised of anti-parallel P-sheets that inserts into the major groove in a similar manner to the DNA recognition domain of I-Ppol (see Galburt and Jurica, this Vol.); (2) a central NUMOD3 domain comprised of a

Fig. 4. Structures of HNH endonucleases bound to dsDNA. 1 Structure of the E9 DNase (2.9 A) bound to double-stranded substrate DNA and Mg2+ (1V14). The E9 DNase HNH motif (black) binds to the minor groove of dsDNA (shown as stick model), inserting the a-helix of the motif and an adjoining loop into the groove and inducing a large structural distortion. This distortion is needed in order for the cleavable phosphodiester bond to be presented to the catalytic metal ion (sphere). 2 Structure of I-Hmul (2.9 A) bound to double-stranded DNA and Mn2+ in the cleaved, product complex (Shen et al. 2004). A 40° bend is produced in the DNA by the insertion of a DNA recognition domain into the minor groove. The Mn2+ ion is adjacent to the cleaved bond. The HNN motif (black) is part of a larger protein with additional domains that contact DNA through contacts to the major and minor grooves (see text for details)

Fig. 4. Structures of HNH endonucleases bound to dsDNA. 1 Structure of the E9 DNase (2.9 A) bound to double-stranded substrate DNA and Mg2+ (1V14). The E9 DNase HNH motif (black) binds to the minor groove of dsDNA (shown as stick model), inserting the a-helix of the motif and an adjoining loop into the groove and inducing a large structural distortion. This distortion is needed in order for the cleavable phosphodiester bond to be presented to the catalytic metal ion (sphere). 2 Structure of I-Hmul (2.9 A) bound to double-stranded DNA and Mn2+ in the cleaved, product complex (Shen et al. 2004). A 40° bend is produced in the DNA by the insertion of a DNA recognition domain into the minor groove. The Mn2+ ion is adjacent to the cleaved bond. The HNN motif (black) is part of a larger protein with additional domains that contact DNA through contacts to the major and minor grooves (see text for details)

pair of a-helices that insert into the minor groove; (3) a C-terminal 'IENR1' domain containing a helix-turn-helix that inserts into the major groove (Sit-bon and Pietrokovski 2003; Shen et al. 2004). The last two domains are similar to those found within the GIY-YIG homing endonuclease I-TevI (see van Ro-ey and Derbyshire, this Vol.). This sharing of DNA recognition modules between HNH, His-Cys and GIY-YIG endonucleases indicates that 'domain exchange' has occurred during the evolution of these families (Shen et al. 2004). Interestingly, I-Hmul makes fewer sequence-specific contacts with DNA than either LAGLIDADG or His-Cys box homing enzymes and accordingly shows weaker sequence specificity, illustrated by its ability to cleave intron-plus and intron-minus genes (Shen et al. 2004).

Members of other homing endonuclease groups (see Chevalier et al., this Vol.) function as dimers with each active site cleaving one of the DNA strands. In cases where the enzyme is monomeric the protein contains two active sites and resembles a fused dimer (e.g. I-Dmol; Galburt and Stoddard 2002). The oligomeric status of HNH/ppa-Me enzymes can be quite variable; enzymes such as CAD are organized as higher order oligomers, sequence-specific enzymes such as I-Ppol are dimers, which permits cleavage of both DNA strands simultaneously, whereas I-Hmul, colicin DNases and I-Cmoel are monomers (Goodrich-Blair and Shub 1996; Galburt et al. 1999; Friedhoff et al. 1999; Drouin et al. 2000; Maté and Kleanthous 2004).

3.2 Biochemical Properties

Cleavage products of the different enzymes in the HNH/ppa-Me group vary from blunt end-products to overhangs of different sizes, with single-strand nicks produced in some cases such as I-Hmul, colicin E9 and the mitochondrial DNA repair protein MutS (Goodrich-Blair and Shub 1996; Pommer et al. 1998; Drouin et al. 2000; Malik and Henikoff 2000). Where single-strand nicking occurs this correlates with the enzyme being monomeric, although simply being monomeric is not sufficient as, for example, I-Cmoel is monomeric but still produces double-strand breaks (Drouin et al. 2000). Nevertheless, the products of phosphodiester hydrolysis by HNH/ppa-Me enzymes are always the same, 5'-phosphates and 3'-hydroxyls (Friedhoff et al. 1999; Widlak et al. 2000; Pommer et al. 2001). Whilst these are probably the most common cleavage products for endonucleases as a whole, formation of the 3'-hydroxyl is essential for the mechanism of homing/insertion of the genetic element encoding the enzyme.

In most cases, HNH/ppa-Me endonucleases have alkaline pH optima (pH 7.5-9), which is suggestive of one or more HNH histidine(s) being in the un-protonated form for catalysis to proceed (Friedhoff et al. 1999; Mannino et al. 1999; Drouin et al. 2000; Pommer et al. 2001; Widlak and Garrard 2001). This is consistent with the absolutely conserved HNH residue acting as a general base, as proposed for I-Ppol (Galburt et al. 1999). Monovalent salts such as NaCl generally inhibit HNH/ppa-Me endonucleases. This may, in part, be due to electrostatic screening of protein-DNA contacts. In the case of I-Ppol, Na+ ions inhibit catalysis by displacing the Mg2+ to form a non-productive I-PpoI-Na+-dsDNA complex (Galburt et al. 1999).

3.3 HNH/ppa-Me Enzymes Require Single Divalent Cations for Activity

HNH/ppa-Me enzymes in common with most endonucleases require divalent cations for catalysis. However, in contrast to the two-cation mechanisms of most restriction enzymes and some homing endonucleases (see Chevalier et al., this Vol.), only one metal ion is required for catalysis (Galburt et al. 1999; Miller et al. 1999; Maté and Kleanthous 2004). The most commonly used metal ion is Mg2+, although many other divalent cations can also substitute, including Ca2+ and various transition metal ions such as Mn2+, Co2+, Cu2+ and Ni2+ (Wittmayer and Raines 1996; Friedhoff et al. 1999; Pommer et al. 1999; Wid-lak and Garrard 2001). The ability to utilize a broad range of metal ions is in contrast to most restriction endonucleases and the LAGLIDADG homing endonucleases (see Chevalier et al., this Vol.). The role of the metal ion (see below and Chevalier et al., this Vol.) is to stabilize the phosphoanion transition state and the 3'OH leaving group of the scissile bond, the latter via an activated water molecule, as proposed for I-Ppol by Galburt et al. (1999).

Some variation exists as to which metal ions will support catalysis in different enzymes of this group. Zn2+, for example, can support catalysis by I-Ppol and the Serratia nuclease but not colicins or I-Hmul (Wittmayer and Raines 1996; Friedhoff et al. 1999; Pommer et al. 1999,2001; Shen et al. 2004). There have been reports of activity with Zn2+ for colicin E7 DNase (Ku et al. 2002) although, in our hands, no colicin DNases exhibit Zn-dependent activity (Pommer et al. 2001; Keeble et al. 2002).

Given that a variety of metal ions support catalysis by HNH/ppa-Me enzymes the question arises to which is the physiological cofactor. Mg2+ is usually considered to be the in vivo metal ion for endonucleases due to its high natural abundance (-0.5 mM) and highest activity with endonucleases (Cowan 1998). This is in contrast to the very low concentrations of available transition metal ions (Changela et al. 2003). The importance of Mg2+ for cleavage of DNA by HNH enzymes, rather than transition metal ions, in vivo has been demonstrated for the colicin DNases. Walker et al. (2002) identified a group of seven active site residues that inactivated Mg2+ activity in vitro of which a subset were still active in the presence of transition metals. However, all mutants were biologically inactive (Walker et al. 2002).

Although Mg2+ is the likely metal ion in vivo for most HNH/ppa-Me endonucleases, the ability of the single Mg2+ ion to bind the enzyme in the absence of DNA varies. For example, Mg2+ can bind to Serratia, T4 endo VII and LtrA in the absence of DNA (Miller et al. 1999; Raaijmakers et al. 2001; San Filip-po and Lambowitz 2002) but not to the colicin E9 DNase or I-Ppol (Pommer et al. 1999; Galburt et al. 2000). The metal ion is also required for binding the DNA in I-Cmoel (Drouin et al. 2000).

4 Structural Analysis of HNH Endonuclease Mediated dsDNA Cleavage

Focusing on the catalytic domains of HNH endonucleases, we describe the structural basis for dsDNA cleavage using the recently solved structures of the substrate (H103A E9-DNase-Mg2+-dsDNA; Fig. 4.1) and product (wild-type I-HmuI-Mn2+-dsDNA; Fig. 4.2) complexes (Maté and Kleanthous 2004; Shen et al. 2004). We also account for the apparent inactivity with Zn2+ ions within the HNH motif.

4.1 Relaxation of Substrate Strain Is Conserved in the Cleavage Mechanisms of HNH/ppa-Me Enzymes

A Mg2+-bound substrate complex structure was obtained for the E9 DNase by mutation of the putative general base HNH residue (H103A), thereby inactivating the enzyme (Walker et al. 2002). A total of -700 A2 of the E9 DNase surface area is buried on binding the dsDNA, with the HNH motif inserting into the minor groove (Maté and Kleanthous 2004). Strikingly, the V-shaped PPa-fold of the active site remains undistorted in the complex. Conversely, in order for the scissile phosphodiester bond of substrate DNA to reach the metal center at the base of the HNH motif, the DNA duplex is distorted (Fig. 4.1). The C-terminal a-helix (containing the HNH residue) lies parallel to the helical axis of the DNA minor groove, with residues preceding the motif and the metal ion inserted into the groove itself. The contacts to the DNA predominantly involve the phosphate backbone and cause a significant bend toward the major groove and concomitant widening of the minor groove (from 5.9 to ~9 Á; Fig. 4.1). In the case of apoE7 bound to dsDNA (Hsia et al. 2004) a similar central distortion is also seen, demonstrating that it is an intrinsic property of the HNH motif. Distortions are also seen for the substrate complexes of I-Ppol (Galburt et al. 1999) and Vvn (Li et al. 2003). In all cases distortion induces strain around the scissile bond. In the complex of the E9 DNase the distortion is brought about by the insertion of the Arg96-Glul00 salt bridge into the minor groove, with Arg96 making a hydrogen bond to the hydroxyl of a thymine on the opposing strand (Maté and Kleanthous 2004). An equivalent salt bridge is observed in Vvn causing similar distortion to the DNA minor groove (Li et al. 2003). The conformation of the DNA in the E9 DNase complex is stabilized by a network of hydrogen bonds between the nucleic acid and three charged residues Arg5, Arg54 and Asp51 that straddle the scissile bond. Consistent with the important roles of Arg5, Arg54, Arg96 and GlulOO in deforming the substrate, alanine mutants at these positions abolish dsDNA cleavage activity (Walker et al. 2002; A.H. Keeble and C. Kleanthous, unpubl. results).

Structural distortions are retained within the product complex of I-Hmul bound to Mn2+ and dsDNA although, in part, this is due to a 40° bend 45 base pairs downstream of the cleaved bond (Fig. 4.2; Shen et al. 2004). However, the DNA around the cleaved bond itself is no longer strained with the 5' phosphate moving away from the 3'OH and adopting a relaxed, tetrahedral geometry (Shen et al. 2004). Relaxation of induced strain in the scissile phos-phodiester upon cleavage has been proposed to be part of the mechanism of DNA cleavage by I-Ppol by Galburt et al. (1999; see also Galburt and Jurica, this Vol.) and has also been reported for Vvn (Li et al. 2003). Therefore, it is likely to be a conserved feature of the cleavage mechanism of all HNH/ppa-Me enzymes.

4.2 Metal Ion Coordination in the DNA-Bound Complex

In the structure of H103A E9 DNase bound to dsDNA and Zn2+, the Zn2+ ion exhibits tetrahedral geometry, made up of three protein ligands, Hisl02, Hisl27 and Hisl31, and a phosphate oxygen atom from the scissile bond (Fig. 5.1). This arrangement is also seen for H103A E9 DNase bound to Zn2+

Fig. 5. The HNH/ppa-Me motif is an adaptable center able to bind both alkaline earth and transition metal ions. 1 H103A E9 DNase-Zn2+-dsDNA complex showing tetrahedral geometry around the metal ion (lV15).The Zn2+ ion is coordinated by three motif histidine residues and a phosphate oxygen atom from the DNA; 2 H103A E9 DNase-Mg2+-dsDNA complex showing distorted octahedral geometry (1V14). The metal ion is coordinated by two motif histidines and two phosphate oxygen atoms from the DNA. Hisl31 disengages from the metal ion and is replaced by a water molecule. The sixth coordination site is presumed to be another water molecule. 3 Wild-type I-HmuI-Mn2+-dsDNA product complex showing distorted octahedral geometry to the metal ion (Shen et al. 2004). The two metal-ligat-ing residues from the enzyme are asparagine and aspartic acid in this HNN motif endonu-clease, equivalent to the two histidines of the colicin E9 DNase

Fig. 5. The HNH/ppa-Me motif is an adaptable center able to bind both alkaline earth and transition metal ions. 1 H103A E9 DNase-Zn2+-dsDNA complex showing tetrahedral geometry around the metal ion (lV15).The Zn2+ ion is coordinated by three motif histidine residues and a phosphate oxygen atom from the DNA; 2 H103A E9 DNase-Mg2+-dsDNA complex showing distorted octahedral geometry (1V14). The metal ion is coordinated by two motif histidines and two phosphate oxygen atoms from the DNA. Hisl31 disengages from the metal ion and is replaced by a water molecule. The sixth coordination site is presumed to be another water molecule. 3 Wild-type I-HmuI-Mn2+-dsDNA product complex showing distorted octahedral geometry to the metal ion (Shen et al. 2004). The two metal-ligat-ing residues from the enzyme are asparagine and aspartic acid in this HNN motif endonu-clease, equivalent to the two histidines of the colicin E9 DNase in the absence of DNA where the phosphate oxygen is replaced by water (Maté and Kleanthous 2004). In each complex the imidazole nitrogen-Zn2+ distances (2.0-2.1 Á) are similar to those of other tetrahedral transition metal sites within proteins. By contrast, the distance between the Zn2+ and the phosphate oxygen is shorter, at 1.9 Á, with similar Zn2+-0 bond distances also seen in the E. coli repair enzyme endo IV (Hosfield et al. 1999).

Two of the four histidines within the HNH motif (Hisl02 and Hisl27) are also coopted to bind the Mg2+ ion in the H103A-E9-Mg2+-dsDNA complex (Fig. 5.2). The octahedral coordination of the Mg2+ ion is clearly very different from the tetrahedral geometry of the Zn2+ ion (Fig. 5.1). Furthermore, the metal ions are displaced relative to each other by ~1Á (Maté and Kleanthous 2004). At the current resolution of the H103A E9 DNase-Mg2+-dsDNA complex (2.9 Á) only 5/6 ligands to the Mg2+ can be identified. Two oxygen atoms of the scissile phosphate (OIP and 03'), together with nitrogen atoms from the Hisl02 and Hisl27, form the equatorial ligands, with one of the axial positions taken by a water molecule (Fig. 5,2). The sixth position is assumed to be another water molecule (Maté and Kleanthous 2004). Octahedral coordination is also observed for the Mn2+ ion within the I-HmuI-dsDNA complex (Fig. 5.3) but here two DNA oxygen atoms contact to the metal ion as equatorial and axial ligands (Shen et al. 2004).

This H103A E9 DNase-Mg2+-dsDNA complex represents one of only two examples in the literature where the coordination shell of a Mg2+ ion in an enzyme active has multiple histidine residues, the other being the F plasmid Tral relaxase (Datta et al. 2003). In this case the Mg2+-N bond distances are in the range 2.3-2.6 Á which are comparable to the distances for Hisl02 and Hisl27 to the Mg2+ ion (2.5 and 2.2 Á, respectively). The octahedral coordination of the metal center is distorted, similar to that seen in the I-PpoI-Mg2+-DNA substrate complex (Galburt et al. 1999), indicating further similarities in the cleavage mechanism between these two ppa-Me endonucleases. In contrast to I-Ppol and Serratia nuclease that contact the bound Mg2+ ion through a single protein side chain, E9 DNase, CAD and Vvn endonucleases use two protein ligands that are superimposable with each other; Asp262 and His308 in CAD, Glu79 and Asnl27 in Vvn, and Hisl02 and Hisl27 in the E9 DNase (Li et al. 2003; Maté and Kleanthous 2004; Woo et al. 2004).

4.3 Mechanism of Mg2+-Dependent Cleavage by HNH Endonucleases and Why Zn2+ Does Not Support Catalytic Activity

Many mechanistic similarities exist between the E9 DNase and I-Ppol including minor groove binding by the ppa-Me motif, a distorted substrate configuration in the DNA-bound complex, a single Mg2+ ion coordinated to the motif, and a strained octahedral geometry around the catalytic metal ion. It is likely therefore that the two enzymes cleave the dsDNA by similar mechanisms. In this mechanism, the HNH histidine residue acts as a general base that activates a water molecule for nucleophilic attack of the scissile phosphodiester bond (this role is taken by His98 in I-Ppol). The Mg2+ ion is coordinated by Hisl02 and Hisl27 in the E9 DNase whereas Asnll9 is the only protein coordination site in I-Ppol (equivalent to Hisl27). The Mg2+ ion nevertheless likely serves similar functions in both, acting to stabilize the phosphoanion transition state and activating a water molecule to protonate the 3'OH leaving group (Galburt et al. 1999). Asimilar mechanism has been proposed for the Serratia nuclease where His89 acts as the general base and Asnll9 stabilizes the phosphoanion transition state and activates a water molecule for protonating the leaving group (Friedhoff et al. 1999). In Serratia, Glul27 makes a water-mediated interaction with the Mg2+ whereas this role is taken by Hisl31 in the E9 DNase (shown in Fig. 5,2 for E9). The roles of these residues are still not clear although they are required for activity since mutations at both reduce activity (Miller et al. 1999; Walker et al. 2002). An equivalent residue is found within the active site of T4 endo VII (Raaijmakers et al. 2001) and I-Ppol (Asnl23). Most HNH/ppa-Me endonucleases use arginines to bind and/or distort substrate DNA, although details differ between enzymes. For example, Arg5 in the E9 DNase binds ground-state DNA but the equivalent residue in I-Ppol (Arg61) binds only within the product complex.

We have been unable to detect Zn2+-catalyzed endonuclease activity for any colicin DNase, which has been difficult to rationalize given that the HNH motif resembles a zinc finger and binds Zn2+ ions with the greatest affinity of all transition metals (Pommer et al. 1999). Moreover, other transition metals such as Ni2+ and Co2+ are active in the motif (Pommer et al. 1998,1999). The current structures provide an explanation for these observations. Hisl31, one of the coordinating histidines to the Zn2+ ion, is displaced by more than 2A in the Mg2+-bound structure (Fig. 5,2). We have proposed previously that transition metal mediated activity with metals such as Ni2+ and Co2+ requires two non-protein-bound coordination sites to the tetrahedral metal ion, one for ligation of the scissile phosphate oxygen, the other for the activation of a water molecule for leaving group stabilization (Pommer et al. 2001). Although no structure for Ni2+ bound to an E9 DNase-dsDNA complex is yet available, an earlier structure for the E9 DNase HNH motif bound with Ni2+ and a phosphate molecule is informative (Kleanthous et al. 1999). Here, again, Hisl31 disengages from the metal ion, in this case taking a position midway between that seen in the Mg2+ and Zn2+ structures. This may allow a water molecule to replace the histidine and so serve as a protonating device for DNA hydrolysis, by a mechanism similar to that proposed for Mg2+-based activity. Consistent with the proposal, rapid reaction kinetics suggests that Zn2+ binding to the E9

DNase induces an additional conformational change relative to Ni2+ binding, which is most likely attributable to Hisl31 binding to the Zn2+ ion, generating a catalytically inert complex (Keeble et al. 2002).

5 Conclusions

HNH endonucleases are part of a widespread group of enzymes whose active sites are built around an evolutionarily conserved ppa-Me structural motif. First described in homing enzymes, HNH endonucleases cleave DNA in a range of biological contexts. Biochemical and structural analyses suggest that HNH/ppa-Me enzymes share a conserved, single metal ion (Mg2+ or Ca2+) cleavage mechanism, with the HNH residue (or an equivalent aspar-agine in HNN enzymes) acting as a metal ligand. The other conserved his-tidine of the motif (HNH) is a general base that activates a hydrolytic water molecule, which then attacks the metal-coordinated scissile phosphodiester bond. By analogy with the mechanism for the His-Cys enzyme I-Ppol (also a PPa-Me endonuclease), the Mg2+ ion in HNH enzymes is proposed to stabilize the pentavalent phosphoanion transition state and activate a water molecule to act as a catalytic acid, enabling protonation of the 3'-hydroxyl leaving group. Notwithstanding our current level of understanding of HNH enzymes and their relationship to the broader group of ppa-Me endonucleases, a number of key questions remain to be resolved: (1) To what extent does substrate strain play a role in catalysis since structures of DNA bound to ppa-Me active sites show highly distorted minor grooves and distorted octahedral geometry to the bound Mg2+ ion? (2) The current structures for DNA and metal ions bound to the substrate complex of the colicin E9 DNase and the product complex of I-Hmul show subtly different DNA coordination chemistries to the metal ion. Are these mechanistically relevant? (3) What are the p-KJs of the different active site residues in their liganded states and are they appropriate for their proposed roles? (4) Do all the residues that contact the phosphate backbone and scissile bond play similar catalytic roles in different HNH/ppa-Me enzymes? For example, Arg5 of the E9 DNase is structurally equivalent to Arg61 of I-Ppol yet appears to contact the ground-state substrate, whereas Arg61 only contacts the product phosphate. (5) Is the mechanism of phosphodiester hydrolysis by HNH/ppa-Me enzymes different when the motif is occupied by a transition metal ion such as Ni2+ or Co2+, as suggested by work on the colicin E9 DNase? (6) Which step (binding and distortion, phosphodiester hydrolysis or product release) is rate-limiting for catalysis by HNH/ppa-Me enzymes? The next few years should resolve many of these issues by which time we will be able to more fully appreciate how adaptable the motif is as a catalytic center for the hydrolysis of phosphodiester bonds.

Acknowledgements. Work on HNH enzymes in C.K.'s lab has been supported by funding from the Biotechnology and Biological Sciences Research Council of the UK and the Wellcome Trust. We would like to thank Charles Heise, Nicholas Housden, Lorna Lancaster and Daniel Walker for critical reading of the manuscript and helpful suggestions. We also acknowledge the work of our collaborators Geoffrey Moore (Norwich) and Richard James (Nottingham) for their valuable contributions to nuclease colicin research.

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