Crystal structure of λ exonuclease in complex with DNA and Ca²⁺

Bacteriophage λ exonuclease (λexo) is a ring-shaped homotrimer that resects double-stranded DNA ends in the 5′-3′ direction to generate a long 3′-overhang that is a substrate for recombination. λexo is a member of the type II restriction endonuclease-like superfamily of proteins that use a Mg²⁺-dependent mechanism for nucleotide cleavage. A previous structure of λexo in complex with DNA and Mg²⁺ was determined using a nuclease defective K131A variant to trap a stable complex. This structure revealed the detailed coordination of the two active site Mg²⁺ ions but did not show the interactions involving the side chain of the conserved active site Lys-131 residue. Here, we have determined the crystal structure of wild-type (WT) λexo in complex with the same DNA substrate, but in the presence of Ca²⁺ instead of Mg²⁺. Surprisingly, there is only one Ca²⁺ bound in the active site, near the position of Mg^A in the structure with Mg²⁺. The scissile phosphate is displaced by 2.2 Å relative to its position in the structure with Mg²⁺, and the network of interactions involving the attacking water molecule is broken. Thus, the structure does not represent a catalytic configuration. However, the crystal structure does show clear electron density for the side chain of Lys-131, which is held in place by interactions with Gln-157 and Glu-129. By combining the K131A-Mg²⁺ and WT-Ca²⁺ structures, we constructed a composite model to show the likely interactions of Lys-131 during catalysis. The implications with regard to the catalytic mechanism are discussed.