Abstract
Although lysines are known to be critical for ligand binding to LDL receptor family receptors, relatively small reductions in affinity have been found when such lysines have been mutated. To resolve this paradox, we have examined the specific binding contributions of four lysines, Lys-253, Lys-256, Lys-270, and Lys-289, in the third domain (D3) of receptor-associated protein (RAP), by eliminating all other lysine residues. Using D3 variants containing lysine subsets, we examined binding to the high affinity fragment CR56 from LRP1. With this simplification, we found that elimination of the lysine pairs Lys-253/Lys-256 and Lys-270/Lys-289 resulted in increases in Kd of 1240- and 100,000-fold, respectively. Each pair contributed additively to overall affinity, with 61% from Lys-270/Lys-289 and 39% from Lys-253/Lys-256. Furthermore, the Lys-270/Lys-289 pair alone could bind different single CR domains with similar affinity. Within the pairs, binding contributions of Lys-270 ≫ Lys-256 > Lys-253 ∼ Lys-289 were deduced. Importantly, however, Lys-289 could significantly compensate for the loss of Lys-270, thus explaining how previous studies have underestimated the importance of Lys-270. Calorimetry showed that favorable enthalpy, from Lys-256 and Lys-270, overwhelmingly drives binding, offset by unfavorable entropy. Our findings support a mode of ligand binding in which a proximal pair of lysines engages the negatively charged pocket of a CR domain, with two such pairs of interactions (requiring two CR domains), appropriately separated, being alone sufficient to provide the low nanomolar affinity found for most protein ligands of LDL receptor family members.
Highlights
Small effects on affinity are seen for seemingly critical lysines of LRP1 ligand receptor-associated protein (RAP)
Our findings support a mode of ligand binding in which a proximal pair of lysines engages the negatively charged pocket of a complement-like repeats (CR) domain, with two such pairs of interactions, appropriately separated, being alone sufficient to provide the low nanomolar affinity found for most protein ligands of LDL receptor family members
We used a novel mutagenesis approach involving elimination of all lysines from the ligand D3 other than those being evaluated for their role in binding to CR56
Summary
Small effects on affinity are seen for seemingly critical lysines of LRP1 ligand RAP. The definitive study on the third domain (D3) of the receptor-associated protein (RAP) that established Lys-256 and Lys-270 as critically important lysines for binding of RAP to LRP1 found reductions in affinity of only 26- and 28-fold, respectively, for K256A and K270E mutations, representing losses of only ϳ17% each in binding energy. Another group found a similar 17% reduction in binding energy for the K256A mutation but only a 12% reduction for a K270A mutation [10]. The finding that Lys-289, alone relatively unimportant, can partially compensate for the loss of Lys-270 establishes the feasibility of lysine substitution and so provides an answer to earlier underestimation of the importance of critical lysines in D3 and perhaps other LRP ligands such as plasminogen activator inhibitor-1
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