DNA Binding to the Splicing and Endonuclease Domains of PISceI

The X-ray structure of the intein PI-SceI in complex with a 36-bp duplex DNA that contains its 31-bp recognition sequence (Fig. 2) is the only structure determined thus far for an intein-associated homing endonuclease in complex with its DNA target (Moure et al. 2002). The interactions of other inteins with their substrates could show similar features, especially those that have a substantial degree of sequence homology, such as the HO endonuclease and other VMA1 inteins which recognize similar sequences. In addition, the cocrys-tal structures of several intron-encoded homing endonucleases have also been determined: the homodimeric I-Crel bound to its 22-bp DNA substrate (Fig. 2) and its product reaction (Jurica et al. 1998; Chevalier et al. 2001), I-Msol (the isoschizomer of I-Crel) bound to a 22-bp substrate (Chevalier et al.

Fig. 2. DNA binding to intein and homing endonucleases. Above DNA complex structures of Pl-Scel, I-Crel, and I-Scel. Pl-Scel and I-Scel show an asymmetric DNA-binding mode. In I-Crel, DNA binding is symmetric. The bending of DNA bound to Pl-Scel is more pronounced than in I-Crel and especially I-Scel. Below DNA recognition sequences for PI-Scel, I-Scel and I-Crel. Essential bases are boxed

Fig. 2. DNA binding to intein and homing endonucleases. Above DNA complex structures of Pl-Scel, I-Crel, and I-Scel. Pl-Scel and I-Scel show an asymmetric DNA-binding mode. In I-Crel, DNA binding is symmetric. The bending of DNA bound to Pl-Scel is more pronounced than in I-Crel and especially I-Scel. Below DNA recognition sequences for PI-Scel, I-Scel and I-Crel. Essential bases are boxed

2003), the monomeric I-Scel bound to a 24-bp substrate (Fig. 2; Moure et al. 2003), and I-Anil, which also functions as a maturase, bound to a 31-bp substrate (PDB entry 1P8K; Bolduc et al. 2003). In this section, we discuss the salient features of DNA recognition by this type of enzyme, including DNA bending and flexibility of the protein-DNA interactions. The comparison of divalent metal binding to the active sites in the high-resolution structures of the symmetric I-Crel and asymmetric I-Scel provides a framework in which we address the catalytic mechanism of homing endonucleases.

3.1 Protein-DNA Contacts Across the Splicing and Endonuclease Domains

The PI-Scel/DNA cocrystal structure (Fig. 2) shows the DNA bound along a large groove that is about 100A long that extends from the splicing domain to the endonuclease domain and covers about three and a half turns of the double helix. This is in agreement with biochemical evidence showing that binding of DNA to both domains is necessary for endonuclease activity (Grindl et al. 1998; He et al. 1998). The splicing domain contacts about 17 bp from +6 to +21 while the endonuclease domain contacts 15 bp from -10 to +5 (the region of the DNA target flanking its cleavage sites). In the splicing domain, the DRR and the 53-70 loop interact with the DNA in the major and minor groove, respectively. The DRR and the 53-70 loop in the splicing domain and the 269284 loop in the endonuclease domain are specific to Pl-Scel, other VMA1 interns, and the HO endonuclease, suggesting that inteins may have developed specificity by acquiring different DNA-binding modules.

Biochemical studies have shown that the splicing domain of Pl-Scel is capable of binding DNA independently (Grindl et al. 1998). In fact, the interactions by residues of the DRR region, most notably Arg 94 and His 170, are critical for DNA binding and their mutation abolishes DNA binding completely (He et al. 1998). In contrast, mutations of residues in the endonuclease domain significantly affect only cleavage activity (Gimble and Wang 1996), which supports a coevolution of the two domains. By acquiring the DRR region and the 53-70 loop, the splicing domain of Pl-Scel evolved DNA-binding activity and simultaneously the endonuclease domain lost the ability to bind DNA independently. It has not been demonstrated that the splicing domain of other inteins bind DNA. In Pl-Pful, which also binds a long DNA substrate of 30 bp, the positively charged stirrup subdomain has been suggested to bind DNA (Ichiyanagi et al. 2000). Intron-encoded homing endonucleases, which lack a splicing domain, provide binding and catalysis in a single domain, resulting in an overall less extensive binding surface (Fig. 2). However, the length of the P-saddle in Pl-Scel is about 40 A, whereas this surface in I-Crel is about 70 A long, supporting the idea that Pl-Scel lost its extended saddle architecture when it became associated with a protein-splicing domain that acquired DNA-binding activity (Gimble et al. 1998).

3.2 Protein Conformational Changes

Homing endonucleases do not undergo large global conformational changes upon DNA binding, but several regions at the protein-DNA interface become ordered or experience conformational changes (Jurica et al. 1998; Moure et al. 2002). The superimposed unbound and DNA-bound structures of Pl-Scel and I-Crel reveal that regions disordered in the unbound structures become ordered only in the presence of DNA. In the DRR of Pl-Scel, the 93-102 loop becomes organized, extending the length of the strands and enabling them to interact with the DNA in the major groove. In the endonuclease domain, the 269-284 loop is also disordered in the unbound structures while in the bound structure the loop is ordered and internally stabilized by two hydrogen bonds. The long P-hairpin loop (369-375) that forms part of the saddle undergoes a hinge-flap motion relative to the ligand-free structure, to interact with the major groove of the DNA. The 53-70 loop that straddles between the two domains moves also in a hinge-flap motion to interact with the DNA in the minor groove. This loop is responsible for domain intercommunication, coupling the binding by the DRR region in the splicing domain to DNA distortion and catalysis in the endonuclease domain (Noel et al. 2004). Disulfide bonds inserted between this P-hairpin loop and the 53-70 loop constrain their flexibility and have been used to switch the enzyme between inactive and active DNA-binding conformations to create an endonuclease that can be turned on and off (Posey and Gimble 2002).

In I-Crel, the most prominent change is that the loop that connects the pi and P2 strands of the saddle adopts a twisted conformation, allowing it to maintain interactions over a long distance of 9 bp. The 138-153 loop, located at the C-terminal end of the protein, also becomes ordered upon DNA binding. Because of the large movements of the regions involved in DNA binding, as well as distortions of the bound DNA (described below), modeling of a canonical DNA to an unbound protein structure might not be an effective way to obtain an accurate picture of the protein-DNA interactions.

3.3 DNA Bending

The bound DNA substrates also experience conformational changes with respect to a canonical B-DNA structure. These conformational changes mirror those of the protein and maximize protein-DNA interactions. The most significant feature of the DNA bound to homing endonucleases is its compression in the minor groove region, which allows for the simultaneous insertion of the top and bottom scissile phosphates into the active sites (see Fig. 3; Ju-rica et al. 1998; Moure et al. 2002, 2003). The narrowing of the minor groove is the result of the combination of deviations from the canonical B-DNA conformation in the form of negative rolls in the active site regions. Since different endonucleases compress the minor groove in different degrees there is no correlation between the overall degree of bending of the DNA and the dimensions of the minor groove at the active sites. For instance, Pl-Scel bends the

l-Scel

Fig. 3. Active site regions of Pl-Scel, I-Crel and I-Scel showing the arrangements of metal ions at the active sites looking down through the minor groove of the DNA. Metal ions are depicted as light colored spheres and are numbered; water molecules are represented as small dark spheres. The scissile phosphates on the DNA are also labeled. In I-Crel, the nucleophilic waters are identified by arrows pointing at the scissile phosphates l-Scel

Fig. 3. Active site regions of Pl-Scel, I-Crel and I-Scel showing the arrangements of metal ions at the active sites looking down through the minor groove of the DNA. Metal ions are depicted as light colored spheres and are numbered; water molecules are represented as small dark spheres. The scissile phosphates on the DNA are also labeled. In I-Crel, the nucleophilic waters are identified by arrows pointing at the scissile phosphates

DNA much more severely (60°) than I-Scel which shows no appreciable substrate bending. However, the minor groove is more compressed in I-Scel than in Pl-Scel (see Fig. 3), resulting in a distance of only 5.5 A between the scissile phosphates in I-Scel as opposed to 9.8 A in Pl-Scel. The less compressed minor groove in Pl-Scel is the consequence of the presence of positive rolls in the immediate downstream region from the active sites that widen the minor groove above standard values (Moure et al. 2002). Another important feature is that the minor groove compression can be symmetric, such as in the homodimeric I-Crel, or asymmetric such as in I-Scel, where the cleavage site of the bottom strand is more buried than that of the top strand (Moure et al. 2003). This feature correlates with differences in the cleavage mechanism of these enzymes (see section 4.2).

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