Residue Gln 138 intercalates into the bound DNA and was suspected of inducing the distortions that ultimately lead to the formation of the CSPS by the center YR step. To test this, the mutant enzyme Q138F HincII was made and analyzed ( Joshi, et al. (2006) J. Biol. Chem. 281, 23852-69). Q138F HincII displayed an altered preference for the three wild type HincII sequences: GTTAAC, GTCGAC, and the non-palindromic GTTGAC/GTCAAC. Where wild type prefers the sequence with TA at the center step over that with CG by a factor of about 6, Q138F HincII prefers it by a factor of 36. The origin of the altered preference is not in DNA binding but in the single turnover DNA cleavage rate, which likely represents the actual rate of DNA cleavage (ie. the chemistry step) by the enzyme. The single turnover rate of DNA cleavage by Q138F HincII is reduced significantly from that of wild type, but reduced more when CG is the center step than when it is TA.

High resolution crystal structures of Q138F HincII with the different DNA sequences show that the distortion of the bound DNA is altered, and depends on the DNA sequence bound (that is, either having TA or CG at the center step). The conformation of the protein is also altered, and depends on the sequence of the DNA bound. Binding of divalent cations such as Ca²⁺ (a mimic for the natural cofactor Mg²⁺, which stimulates binding without confering DNA cleavage activity) also alters the conformations of the HincII enzyme and the bound DNA, and differently depending on the DNA sequence bound.

In order to understand how structure influences enzymatic reaction rates, the mechanism of DNA cleavage by HincII (Etzkorn & Horton (2004)Biochemistry 43, 13256, Etzkorn & Horton (2004) J. Mol. Biol. 343, 833) must first be understood. Two metal ions (blue circles, below) bind in the active site and ligate the scissile phosphate. One metal ion activates a ligated water molecule for attack on the phosphorus atom, the other metal ion stabilizes the O3' leaving group. The active site lysine forms a salt bridge with the scissile phosphate stabilizing the additional negative charge which occurs in the transition state. A neighboring phosphate may act as the proton acceptor from the water molecule as it is activated by becoming hydroxide. When these atoms: the attacking water, lysine 129, the neighboring phosphate, the scissile phosphate, metal ions, are in the optimal positions, the reaction is fastest. Misalignments in positioning of any of these groups presumably leads to reduced cleavage rates. Note: when Ca²⁺ is used in the structural studies, only one of the two metal ion binding sites is occupied. In order to view the structure pior to DNA cleavage, Ca²⁺ is used rather than Mg²⁺, and therefore only the one metal ion is visible.

Below is a morph of wild type and Q138F HincII bound to DNA containing C₃G₄ at the center step showing how the mutation Q138F alters the protein-DNA interface, leading to misalignments in the active site, and ultimately slower DNA cleavage kinetics. Mutation of glutamine 138 to phenylalanine results in the F138 side chain sitting differently in the intercalated DNA. It is shifted in order to stack onto the neighboring bases. The shift of the side chain also shifts the main chain, causing neighby alanine 137 to flip away from the DNA. In the wild type structure, the methyl group of ala 137 forms a van der Waals interaction with the sugar ring of a nucleotide in the DNA. Once flipped away, the sugar ring relaxes to a more common pucker (C2' endo). The chain in sugar pucker causes alterations in the sugar-phosphate backbone which propagate several nucleotides away, causing the O5' of the adenine to jut towards the protein near residue 130. The side chain of threonine 130 avoids a steric clash with Ade O5' by shifting away from the DNA. The active site lysine, lysine 129, neighbors threonine 130, and is also shifted. The shifting of the protein at 130-127 (residue 127 is one of the aspartates which ligates the metal ion (cyan sphere)) results in misalignments in the active site which are likely responsible for the reduced DNA cleavage kinetics.

Greater misalignments occurr in the active sites when DNA containing C₃G₄ is bound than DNA containg TA. In addition, there is greater disruption of the CSPS in the structure with T₃A₄ than with C₃G₄. Below is a morph between Q138F HincII bound to CG and that bound to T₃A₄. The weaker stacking energy of the two adenines allows the DNA to distort to compensate for misalignments at the protein-DNA interface created by the mutation at residue 138. The guanine bases in the C₃G₄ containing DNA hold on to the CSPS, and the misalignments are then felt most in the active site. (Note: center step purine bases, A in T₃A₄ and G in C₃G₄ are shown in dark blue below)

A full understanding of the relationship between the mutation, Q138F, and the altered sequence preference came with the structure of Q138F HincII bound to DNA and Mn²⁺ (below). The CSPS (dark blue bases) must pull apart (grey arrow) to pull apart the neighboring phosphate and allow space for the nucleophilic water (grey sphere) to bind at the metal ion (yellow sphere). This must occur regardless of which sequence is bound, but occurs more frequently with the TA center step due to the weaker stacking energy of adenine relative to guanine.

Hence, the alteration in specificity of HincII by the mutation Q138F is derived from the differences in the base stacking energetics of the center step purines. Taking the rate constants for cleavage of CG vs. TA by Q138F, the difference in the cross-strand stacking energies of GG vs. AA is approximately 1.0 kcal/mole.

Origin of DNA Distortion in HincII

Horton Lab Homepage
Department of Biochemistry and Molecular Biophysics
University of Arizona
nhorton@u.arizona.edu
http://www.biochem.arizona.edu/horton/index.html

January 11, 2008

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