Unfolding of the RAP-D3 Helical Bundle Facilitates Dissociation of RAP−Receptor Complexes

Abstract

The receptor-associated protein (RAP) functions as an escort protein for receptors of the low-density lipoprotein receptor (LDLR) family by preventing premature intracellular binding of ligands and assisting with delivery of mature receptors to the cell surface. The modulation of affinity by pH is believed to play an important role in the escort function of RAP, because RAP binds tightly to proteins of the LDLR family at near-neutral pH early in the secretory pathway where its high affinity precludes premature binding of ligands but then dissociates from bound receptors at the lower pH of the Golgi compartment. The third domain of RAP (RAP-D3), which forms a three-helix bundle, is sufficient to reconstitute the escort activity. Here, we test the hypothesis that low-pH induced unfolding of the RAP-D3 helical bundle facilitates dissociation of RAP-receptor complexes. First, variants of RAP-D3 resistant to low pH-induced unfolding were constructed by replacing interior histidine residues with phenylalanines. In contrast to native RAP-D3, which exhibits an unfolding pKa of 6.3 and a Tm of 42 degrees C, the most hyperstable variant of RAP-D3, in which four histidine residues are replaced with phenylalanine, has an unfolding pKa of 4.8, and a Tm of 58 degrees C. The phenylalanine substitutions in RAP-D3 confer increased stability to pH-induced dissociation of complexes formed between RAP-D3 and a two-repeat fragment of the LDLR (LA3-4). When introduced into full-length RAP, the four mutations that confer hyperstability on RAP-D3 interfere with transport of endogenous LRP-1 to the cell surface in a dominant negative fashion under conditions where expression of normal RAP has no effect on LRP-1 transport. Our studies support a model in which low pH-dependent unfolding of RAP-D3 facilitates dissociation of RAP from the LA repeats of LDLR family proteins in the mildly acidic pH of the Golgi.

Generation of a Potent Low Density Lipoprotein Receptor-related Protein 1 (LRP1) Antagonist by Engineering a Stable Form of the Receptor-associated Protein (RAP) D3 Domain

“…3, A and B). The RAP D3 Quad H:F mutant melted at 58°C, nearly 15°C higher than that of the wild type protein and remarkably similar to the melting point (T m ) of 57.6°C reported by Estrada et al (11). In contrast, our RAP D3 disulfide mutant remained folded until a substantially higher temperature with a T m of 65°C.…”

“…1A). To serve as a control for this mutant, we also altered four of the histidine residues (His-257, His-259, His-268, and His-290) into phenylalanines (RAP D3 Quad H:F) to eliminate the "histidine switch" that has previously been demonstrated (10,11) to increase the stability of the RAP D3 domain in low pH (Table 1 and Fig. 1B).…”

“…This structure revealed a three-helical bundle domain consisting of a short helix of 10 amino acids and two longer helices containing 40 and 38 amino acids, respectively. Importantly, the work identified a histidine switch in which the low pH environment of the Golgi results in protonation of histidine residues, resulting in an unfavorable accumulation of positive charges between helices 2 and 3, causing the domain to unfold and dissociate from LRP1 (10,11). This D3 destabilization model in which dissociation occurs due to pH-mediated denaturation of D3 has recently been questioned by Jensen et al (31).…”

“…Lowering of the pH activates a histidine switch in which protonation of histidine residues on the second and third helices of RAP D3 destabilizes this domain, leading to its denaturation (10,11). Mutation of key histidine residues generates a molecule with increased stability at low pH that does not dissociate from LRP1 and fails to release from LRP1 in the Golgi, resulting in ineffective delivery of LRP1 to the cell surface due to retrieval of the receptor-RAP D3 complex from the Golgi back to the ER (10,11).…”

A proximal pair of positive charges provides the dominant ligand-binding contribution to complement-like domains from the LRP (low-density lipoprotein receptor-related protein)

“…Whereas RAP binds with low nanomdar affinity to many members of the LDLR family [29], it binds much less tightly to LDLR itself [30]. Consistent with this lower affinity for the intact receptor, the K d value for the interaction of the two domain fragment of LDLR, LB3–LB4, with domain D3 of RAP is ~1 μM at I =0.15 [31]. The low binding affinity seems surprising given that LB3 and LB4 are the two LDLR domains that each contains the DxDxD motif and the D3 domain of RAP contains two critical lysine residues (Lys 256 and Lys 270 ) that have been implicated in binding to LRP [32], each of which occurs as a part of a proximal pair of lysine residues (Lys 256 with Lys 253 and Lys 270 with Lys 289 ).…”