Supporting Information

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Fig. 4. Isothermal titration curves. (A) The titration curve of hEC1 against Y369S demonstrates the association to be exothermal. 8 ml aliquots of Y369S (0.43 mM) were successively injected into a 0.04 mM solution of hEC1. The association affinity constant and the binding enthalpy were derived by curve fitting using the single set of independent sites model. (B) Titrating hEC1 (0.035 mM) against G194S+S (0.66 mM) using 5-ml injections reveals an unusual endothermal association reaction. Both titrations yield a binding stoichiometry of 1.0 ± 0.1.

Fig. 5. Schematic representation of local rearrangements in InlA-variants. Leucine-rich-repeat (LRR) 5-7 of InlA-variants are shown in green, interacting residues of hEC1 in yellow. Amino acid substitutions are highlighted in red. (A) InlA/hEC1: disordered Ser-192_InlA and its water-mediated interactions to Phe-17_hEC1-O and Pro-18_hEC1-O. Two residues of neighboring repeats, Asp-213_InlA and Ser-172_InlA, additionally stabilize the water cluster near Ser-192_InlA. The shortened LRR6 excludes Asn-195_InlA from the canonical asparagine ladder within the hydrophobic core and creates a large depression facing Glu-54_hEC1 and Lys-61_hEC1. (B) Substituting Ser-192_InlA by asparagine creates a direct hydrogen bond to Phe-17_hEC1 by excluding one water molecule previously stabilized by Ser-172_InlA. (C) Restoring the canonical LRR architecture by inserting a serine after position 194 and replacing Gly-194_InlA by serine flips Asn-195 into the hydrophobic core to complete the asparagine ladder and reduces the distance to Glu-54_hEC1 and Lys-61_hEC1. (D) Combination of substitutions S192N and G194S+S (B and C). The restored LRR6 allows Asn-192_InlA to form an intramolecular hydrogen-bond to Ser194_InlA-N while retaining the direct hydrogen bond to Phe-17_hEC1. As a result, Asn-192 no longer excludes a second water molecule between InlA and hEC1 (see B), weakening the interaction. A shorter hydrogen bond from Glu-54_hEC1 to bridging water molecules may indicate tighter interaction with the inserted serine +S. Alternatively, these shorter distances may result from a different crystal packing, because overall tighter binding is not observed thermodynamically. In S192N-G194S+S/hEC1 and G194S+S-Y369S/hEC1, both of which crystallized in space group P1, SI Table 1, Arg-55_hEC1 of hEC1 forms a hydrogen bond to LRR7-10 of a symmetry-related molecule, possibly shifting Glu-54_hEC1 toward InlA.