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Integrative structural analysis defines how LRPAP1 modulates ligand binding in LRP2

ABSTRACT: The low density lipoprotein receptor-related protein 2 (LRP2 or megalin) is a multiligand endocytic receptor implicated in the homeostasis of several organs. Mutations in the LRP2 gene are associated with severe systemic disease. It is well-know that the low density lipoprotein receptor-related protein-associated protein 1 (LRPAP1 or RAP) interacts with LRP2 modulating its function. A single copy of LRPAP1 was shown to interact with complement-type repeats in LRP2, however this domain is highly redundant in LRP2, hinting at a different stoichiometry of the complex. Here, using cryogenic-electron microscopy, AlphaFold and cross-linking mass spectrometry, we provide structural insights into human recombinant LRP2 in the presence and absence of LRPAP1. Our integrative approach reveals three additional LRPAP1 sites in LRP2. Some of these LRPAP1 binding regions overlap with ligand binding sites. This finding, supported by competitive in vitro binding assays supports the hypothesis that LRPAP1 acts as a direct modulator of LRP2's ligand-binding activity. In addition, we identify several pathogenic LRP2 mutations located at the LRP2-LRPAP1 interface and LRPAP1 binding regions unique to LRP2 within the LRP family. Taken together, our study provides a broader molecular landscape of LRP2-LRPAP1 interactions, offering insights into its clinical and functional role.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Andrea Graziadei

LAB HEAD: Andrea Graziadei

PROVIDER: PXD066532 | Pride | 2026-03-21

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	250122_KR_Megalin_Rap_SDA_AT_TA.conf	Other
	250122_RAP-Megalin_exact_constructs_plus_top600.fasta	Fasta
	250131_KR_Meg_Rap_SDA_AT_TA_5pc_links_boost_btw_Links_xiFDR2.3.2.csv	Csv
	250131_KR_Meg_Rap_SDA_AT_TA_5pc_links_boost_btw_noncov.mzid	Mzid
	E241205_13_HT_AD_120_KR_Meg_Rap_AspN_Trypsin_SDA_A5_inj1.mgf	Mgf

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Project description:[NiFe]-hydrogenases catalyse the reversible splitting of hydrogen. The catalytic metal centre (NiFe(CN)2CO) is unique in biology, and assembled by an intricate protein machinery, in a process that is still being explored. We hypothesised a structural, ATP-dependent, mechanistic explanation for the assembly of the Fe(CN)2CO fragment via the HypCD complex. We carried out a crosslinking mass spectrometry (crosslinking MS) analysis to study the structure of the HypCD complex, both with (holoprotein) and without (apoprotein) the metal cofactor, and both with and without the addition of ATP. We used the UV-photoactivatable crosslinking reagent sulfo-SDA, which has been shown to have excellent performance when studying dynamic and flexible protein complexes. For photoactivation we used a high-powered LED which enabled the use of exceptionally short reaction times (20 seconds) and gave strikingly clear results. From the resulting crosslinked residue pair patterns identified, we were able to unambiguously distinguish between holoprotein with and without ATP. Crosslinks found in holoprotein, in the absence of ATP, suggested a “closed” protein conformation. When the HypCD holoprotein was crosslinked in the presence of ATP, two distinct crosslink bands were almost entirely absent, indicating that the protein conformation had shifted to an “open” conformation. Interestingly, no shift in protein conformation was evident in the crosslinked apoprotein, which implied that the cofactor was central to protein conformational dynamics. Crosslinking MS data helped to explain, and was in agreement with, protein structures predicted by AlphaFold2 (which were subsequently refined by density functional theory (DFT) modeling for placing the cofactor). Considering all the experimental data from this study led to the conclusion that the binding of ATP alone, not its hydrolysis, is required for the transfer of the Fe(CN)2CO fragment to the apo-hydrogenase large subunit.

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Integrative structural analysis defines how LRPAP1 modulates ligand binding in LRP2

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