Download Exonuclease active site: a more complete description

Exonuclease active site: a more complete description
The structures of phi29 DNAP containing ssDNA and several complexes of
Klenow fragment (Beese and Steitz, 1991; Brautigam et al., 1999) were superimposed
using their respective exonuclease domains, allowing us to tentatively assign the position
of the nucleophilic water in the phi29 structure. The metal ion binding sites in phi29
have been mutated (D12A/D66A) (Bernad et al., 1989) to allow formation of a stable
substrate complex. Due to these mutations and to coordination geometry constraints, the
atoms occupying these sites cannot be unambiguously identified as metal ions and
consequently they have been modeled as water molecules. We also observe a water
molecule bound at position that we have identified as the location of the nucleophilic
water based on these superpositions. As expected, the distances between the water
molecules and the ligands are, in general, larger than they would be for metals (Figure
S1).
While the overall interface between protein and substrate in both copies of
polymerase in this crystal form can be described by similar sequence non-specific
hydrophobic and hydrogen bonding interactions, the detailed interactions at the active
sites are significantly different. In copy A, Y165, a residue in the conserved exonuclease
III motif (Bernad et al., 1989), and K143 (de Vega et al., 1997) are solvent exposed. In
copy B, two conformations are observed in the maps: (1) the conformation observed for
copy A is very poorly ordered in copy B because the crystal contacts stabilizing it in copy
A are missing near copy B, and (2) a conformation in which Y165 and K143 participate
in the exonuclease active site: the hydroxyl of Y165 interacts with the presumed water
nucleophile (Beese and Steitz, 1991) as well as with K143, whose -amine group
interacts with the hydroxyl of Y165, with the catalytic aspartate of the exonuclease III
motif (D169), and with the scissile phosphate through a water-mediated hydrogen bond
(Figure S1). The quality of the maps for the former conformation of copy B is extremely
poor due to disorder, so only the more ordered conformation is included in the model.
Although it was not obvious from structure-based alignments (Kamtekar et al.,
2004) that K143 had a structural homolog in the non-protein-primed members of the
polymerase B-family, it had been shown to be critical in the exonuclease reaction in
phi29 DNA polymerase (de Vega et al., 1997), suggesting that there is a functionally
homologous residue in non-protein-primed B-family DNA polymerases. Indeed,
inspection of the RB69 DNA polymerase exonuclease structure reveals that, in the
context of a complete active site, if the exonuclease III motif tyrosine (Y323) were to flip
into the active site to stabilize the nucleophile, as biochemical (Brautigam and Steitz,
1998; Wang et al., 2004) and crystallographic studies (Wang et al., 1997) have suggested,
one would expect that K302 of RB69 DNA polymerase would be, as predicted by de
Vega et al. (1997), the functionally homologous residue to K143 of phi29 DNA
polymerase.
These results demonstrate that there are two stable conformations at the
exonuclease active site of B-family DNA polymerases, a hypothesis previously proposed
from comparisons of T4 and RB69 DNA polymerase exonuclease structures with the
Klenow fragment exonuclease structure (Beese and Steitz, 1991; Wang et al., 2004;
Wang et al., 1996). In one conformation, the tyrosine from the exonuclease III motif is
solvent exposed, while in the other conformation, the tyrosine interacts with the scissile
phosphate through the nucleophile, and a conserved lysine stabilizes the catalytic
aspartate of the exonuclease III motif, consistent with biochemical results (de Vega et al.,
1997). The latter conformation seems to be the more chemically and biologically
relevant complex for exonuclease activity.
Furthermore, since the participation of the
conserved tyrosine in the exonuclease active site constitutes a necessary event during the
exonucleolytic cycle (de Vega et al., 1997), the two conformations observed suggest that
the movement of the conserved tyrosine and lysine residues into the active site sets up the
active site for the exonucleolysis reaction in the B-family of DNA polymerases.
Figure S1
The exonuclease active site of a superposition of both copies of polymerase from the
single stranded DNA crystal form. Aligned on the exonuclease domain, copy A is beige
and copy B is grey, and the single stranded DNA substrates are orange and dark grey,
respectively. The K143, Y165, and the catalytic aspartate residues that are not mutated to
alanines are shown in stick representation. Green arrows demonstrate the movement of
Y165 and K143 from the open conformation to the closed conformation. The proposed
nucleophilic water is represented as a blue sphere, and the waters occupying the metal ion
binding sites are indicated. The black dashed lines are hydrogen bonds that demonstrate
the interactions between K143 and Y165 with each other and with other parts of the
active site. The interactions between the waters in the metal binding sites and the protein
are represented as gray dashes. Most of the interactions that the water in the metal ion B
site would be making with the protein are missing due to the D12A/D66A mutations in
the polymerase used in these studies. The distance between the nucleophile and the water
near the metal ion A binding site is represented as a yellow dash. This distance is longer
than it would be if a metal ion were in this site.
Figure S2
A schematic representation of protein-DNA interactions from one copy of the ternary2
complex. The interactions are generally maintained in the other copy of the ternary2
complex, and in the ternary1 and binary complexes. The base of each nucleotide is
represented by a rectangle and the phosphate and sugar of a nucleotide are represented by
a circle. The incoming dNTP is indicated in yellow. The residues are color coded
according to interaction type: red, direct interaction with base; orange, hydrophobic
interaction; green, water-mediated interaction; pink, interaction through a water network;
grey, metal-mediated interaction; blue, direct interaction with sugars and phosphates.
Asterisks indicate the nucleotides with which conserved waters interact.
Movie
This movie illustrates the replication cycle of phi29 DNAP based on the structures of the
binary complex (copy A) and ternary complex (ternary2, copy A). Since the insertion
site is unavailable when the fingers are opened, the incoming dNTP was modeled as
initially binding the conserved lysines of the fingers subdomain in the opened binary
complex. As the fingers close, the conserved tyrosine residues (Y254, Y390; represented
as spheres) move out of the insertion site so that the incoming dNTP can interact with the
templating base in the nascent base pair binding pocket. In accordance with the
precedent established by structural studies with the T7 RNAP elongation cycle (Yin and
Steitz, 2004), the active site remains relatively unchanged during the chemical step and
the fingers open upon pyrophosphate dissociation in this animation. This movie
highlights the proposed movement of the conserved tyrosine residues during the
conformational change of the fingers subdomain, and the concomitant role of the
conserved tyrosine residues in B-family translocation. Morph (Echols et al., 2003; Krebs
and Gerstein, 2000) was used to generate intermediates between the various steps of the
cycle. Images were made in Pymol (DeLano, 2002) and the movie was compiled in
VideoMach 4.0.1.
References
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