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An Evaluation of the Crystal Structure of C-terminal Truncated Apolipoprotein A-I in Solution Reveals Structural Dynamics Related to Lipid Binding

ABSTRACT: Apolipoprotein (apo)A-I mediates many of the anti-atherogenic functions attributed to high density lipoprotein (HDL). Unfortunately, efforts toward a high-resolution structure of full-length apoA-I have not been fruitful, though there have been successes with deletion mutants. Recently, a C-terminal truncation (apoA-IΔ185-243) was crystallized as a dimer. The structure showed two helical bundles connected by a long, curved pair of swapped helical domains. To compare this structure to that existing under solution conditions, we applied small angle X-ray scattering and isotope-assisted chemical cross-linking to apoA-IΔ185-243 in its dimeric and monomeric forms. For the dimer, we found evidence for the shared domains and aspects of the N-terminal bundles, but not the molecular curvature seen in the crystal. We also found that the N-terminal bundles equilibrate between open and closed states. Interestingly, this movement is one of the transitions proposed during lipid binding. The monomer was consistent with a model in which the long shared helix doubles back onto the helical bundle. Combined with the crystal structure, these data offer an important starting point to understand the molecular details of HDL biogenesis.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Diogo Borges Lima

LAB HEAD: Paulo C Carvalho

PROVIDER: PXD006574 | Pride | 2017-05-30

REPOSITORIES: Pride

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An Evaluation of the Crystal Structure of C-terminal Truncated Apolipoprotein A-I in Solution Reveals Structural Dynamics Related to Lipid Binding.

Melchior John T JT Walker Ryan G RG Morris Jamie J Jones Martin K MK Segrest Jere P JP Lima Diogo B DB Carvalho Paulo C PC Gozzo Fábio C FC Castleberry Mark M Thompson Thomas B TB Davidson W Sean WS

The Journal of biological chemistry 20160111 10

Apolipoprotein (apo) A-I mediates many of the anti-atherogenic functions attributed to high density lipoprotein. Unfortunately, efforts toward a high resolution structure of full-length apoA-I have not been fruitful, although there have been successes with deletion mutants. Recently, a C-terminal truncation (apoA-I(Δ185-243)) was crystallized as a dimer. The structure showed two helical bundles connected by a long, curved pair of swapped helical domains. To compare this structure to that existin ...[more]

PMID: 26755744

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Project description:DM64 is a toxin-neutralizing serum glycoprotein isolated from the South American opossum Didelphis aurita. This ophiophagous marsupial is naturally resistant to snake envenomation and has evolved such biochemical defense as a trophic adaptation. The endogenous antitoxin is a human α1B-glycoprotein homolog of 64 kDa, specifically targeting myotoxic phospholipases A2, which account for most local tissue damage associated with viper snakebites. This study investigated the noncovalent complex formed between native DM64 and myotoxin II, a myotoxic calcium-independent phospholipase-like protein from Bothrops asper venom. Analytical ultracentrifugation, size exclusion chromatography, and small-angle X-ray scattering (SAXS) data indicated that DM64 is monomeric in solution and binds equimolar amounts of the toxin. Initial attempts to solve the structure of DM64 experimentally failed due to technical limitations caused by N-glycan heterogeneity and low recovery of heterologous protein. Classical molecular modeling techniques were impaired by the lack of templates with more than 25% sequence identity with DM64. An integrative structural biology approach was then applied to generate a refined three-dimensional model of the inhibitor bound to myotoxin II. The five immunoglobulin-like domains of DM64 were individually modeled using the I-TASSER suite. Distance constraints generated by cross-linking mass spectrometry of the complex guided the docking of DM64 domains to the crystal structure of myotoxin II (1CLP), using the Rosetta docking suite. The model of the DM64-myotoxin II complex was successfully fitted to a SAXS envelope. Inter-protein cross-links and limited hydrolysis analyses shed light on the inhibitor's regions involved with toxin interaction, revealing the critical participation of the first, third, and fifth domains of DM64. Our data showed that the fifth domain of DM64 binds to myotoxin II’s amino-terminal and -wing regions. The third domain of the inhibitor acts in a complementary way to the fifth domain. Their binding to these toxin regions presumably interferes with dimerization, a critical step in the allosteric activation of myotoxic PLA2-like proteins. Additionally, the first domain of DM64 likely interacts with the functional site of the toxin putatively associated with membrane anchorage. We propose that both mechanisms concur to effectively inhibit myotoxin II toxicity by DM64 binding. In summary, we argue such topological characterization of this toxin-antitoxin complex to constitute an essential first step toward the rational design of novel peptide-based antivenom therapies targeting snake venom myotoxins.

			Action	DRS
	Data071114_03.wiff	Wiff
	Data071114_03.wiff.1.idx2	Wiff
	Data071114_03.wiff.scan	Wiff
	Data071114_05.wiff	Wiff
	Data071114_05.wiff.1.idx2	Wiff