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. Author manuscript; available in PMC: 2009 May 2.
Published in final edited form as: J Mol Biol. 2008 Mar 4;378(3):653–665. doi: 10.1016/j.jmb.2008.02.058

Figure 3. Construction and substrates of the designed Cre heterotetramer.

Figure 3

(a) “Spatial matching” of recombinase and substrates. The alternating ABAB arrangement of CreWT and CreALSHG subunits can be enforced by using two “orthogonal” interfaces surfaces, thereby favoring recombination of substrate pairs containing alternating 13 bp repeat arrangements (left) over un-matched arrangements (middle and right).

(b) “Split interface” variants”. CreAA and ALSHG-F contain complimentary combinations of orthogonal CreAAF and CreWT interfaces with CreWT and CreALSHG DNA specificities. The Ala334-Phe substitution is indicated by the triangular CTH representation and the Met299-Ala/Val304-Ala CTD substitutions are indicated by the wedge-shaped indentation, while the corresponding CreWT interfaces are indicated by the circle and circular indentation.

(c) CTH/CTD Cre-Cre interface. In the CTD (chain B of 1CRX, green), residues Met299, Ala302 and Val304 form a hydrophobic docking surface for residues Ala334, Met335 and Leu338 from the CTH (A chain, purple) from the adjacent subunit. Positions 299, 304 and 334 are the focus of this study and are highlighted in yellow.

(d) Interface residues targeted for engineering. CTD residues Met299 and Val304 form a shallow pocket that accommodates CTH-residue Ala334 from the adjacent subunit (left panel). A hypothetical un-minimized model of the “size-switch” CreAAF variant is shown in the right panel. The Ala299 and Ala304 side-chain truncations form a larger pocket to accommodate the larger Phe334. This volume swap creates alternate interfaces surfaces that interact unfavorably with CreWT surfaces. Contacts between CreAAF and CreWT subunits are disfavored because cavity formation or steric clashes would result.