Unique structural features of the AIPL1–FKBP domain that support prenyl lipid binding and underlie protein malfunction in blindness

FKBP-domain proteins (FKBPs) are pivotal modulators of cellular signaling, protein folding, and gene transcription. Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a distinctive member of the FKBP superfamily in terms of its biochemical properties, and it plays an important biological role as a chaperone of phosphodiesterase 6 (PDE6), an effector enzyme of the visual transduction cascade. Malfunction of mutant AIPL1 proteins triggers a severe form of Leber congenital amaurosis and leads to blindness. The mechanism underlying the chaperone activity of AIPL1 is largely unknown, but involves the binding of isoprenyl groups on PDE6 to the FKBP domain of AIPL1. We solved the crystal structures of the AIPL1–FKBP domain and its pathogenic mutant V71F, both in the apo form and in complex with isoprenyl moieties. These structures reveal a module for lipid binding that is unparalleled within the FKBP superfamily. The prenyl binding is enabled by a unique “loop-out” conformation of the β4-α1 loop and a conformational “flip-out” switch of the key W72 residue. A second major conformation of apo AIPL1–FKBP was identified by NMR studies. This conformation, wherein W72 flips into the ligand-binding pocket and renders the protein incapable of prenyl binding, is supported by molecular dynamics simulations and appears to underlie the pathogenicity of the V71F mutant. Our findings offer critical insights into the mechanisms that underlie AIPL1 function in health and disease, and highlight the structural and functional diversity of the FKBPs.

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Elucidating the structural features of ABCA1 in its heterogeneous membrane environment.

10.1101/2021.05.23.445323 ◽

2021 ◽

Author(s):

Sunidhi S ◽

Sukriti Sacher ◽

Parth Garg ◽

Arjun Ray

Keyword(s):

Conformational Changes ◽

Transmembrane Protein ◽

Direct Interaction ◽

Structural Features ◽

Lipid Binding ◽

Lipid Monolayer ◽

Alternative Theory ◽

Lipid Dynamics ◽

Lipid Environment ◽

Dynamics Simulations

ABCA1 plays an integral part in Reverse Cholesterol Transport (RCT) and is critical for maintaining lipid homeostasis. One theory of lipid efflux by the transporter (alternating access) proposes that ABCA1 harbors two different conformations that provide alternate access for lipid binding and release, leading to sequestration via a direct interaction between ABCA1 and its partner, ApoA1. The alternative theory (lateral access) proposes that ABCA1 obtains lipids laterally from the membrane to form a temporary extracellular reservoir containing an isolated pressurized lipid monolayer caused by the net accumulation of lipids in the exofacial leaflet. Recently, a full-length Cryo-EM structure of this 2,261-residue transmembrane protein showed its discreetly folded domains and conformations, as well as detected the presence of a tunnel enclosed within ECDs. While the tunnel was wide enough at the proximal end for accommodating passage of lipids, the distal end displayed substantial narrowing, making it inaccessible for ApoA1. Therefore, this structure was hypothesized to substantiate the lateral access theory, whereby ApoA1 obtained lipids from the proximal end. Utilizing long time-scale multiple replica atomistic molecular dynamics simulations (MDS), we simulated the structure in a heterogeneous lipid environment and found that along with several large conformational changes, the protein widens enough at the distal end of its ECD tunnel to now enable lipid accommodation. In this study we have characterized ABCA1 and the lipid dynamics along with the protein-lipid interactions in the heterogeneous environment, providing novel insights into understanding ABCA1 conformation at an atomistic level.

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Structural features and lipid binding domain of tubulin on biomimetic mitochondrial membranes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1619806114 ◽

2017 ◽

Vol 114 (18) ◽

pp. E3622-E3631 ◽

Cited By ~ 18

Author(s):

David P. Hoogerheide ◽

Sergei Y. Noskov ◽

Daniel Jacobs ◽

Lucie Bergdoll ◽

Vitalii Silin ◽

...

Keyword(s):

Electrochemical Impedance ◽

Physiological Role ◽

Structural Features ◽

Lipid Binding ◽

Binding Domain ◽

Water Soluble ◽

Mitochondrial Outer Membrane ◽

Mitochondrial Membranes ◽

Dynamics Simulations ◽

Integral Proteins

Dimeric tubulin, an abundant water-soluble cytosolic protein known primarily for its role in the cytoskeleton, is routinely found to be associated with mitochondrial outer membranes, although the structure and physiological role of mitochondria-bound tubulin are still unknown. There is also no consensus on whether tubulin is a peripheral membrane protein or is integrated into the outer mitochondrial membrane. Here the results of five independent techniques—surface plasmon resonance, electrochemical impedance spectroscopy, bilayer overtone analysis, neutron reflectometry, and molecular dynamics simulations—suggest that α-tubulin’s amphipathic helix H10 is responsible for peripheral binding of dimeric tubulin to biomimetic “mitochondrial” membranes in a manner that differentiates between the two primary lipid headgroups found in mitochondrial membranes, phosphatidylethanolamine and phosphatidylcholine. The identification of the tubulin dimer orientation and membrane-binding domain represents an essential step toward our understanding of the complex mechanisms by which tubulin interacts with integral proteins of the mitochondrial outer membrane and is important for the structure-inspired design of tubulin-targeting agents.

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Interaction of Aryl Hydrocarbon Receptor-interacting Protein-like 1 with the Farnesyl Moiety

Journal of Biological Chemistry ◽

10.1074/jbc.m113.476242 ◽

2013 ◽

Vol 288 (29) ◽

pp. 21320-21328 ◽

Cited By ~ 23

Author(s):

Anurima Majumder ◽

Kota N. Gopalakrishna ◽

Pallavi Cheguru ◽

Lokesh Gakhar ◽

Nikolai O. Artemyev

Keyword(s):

Aryl Hydrocarbon Receptor ◽

Solution Structure ◽

Structural Model ◽

Mutational Analysis ◽

Normal Function ◽

Severe Form ◽

Scattering Data ◽

Interacting Protein ◽

Fk506 Binding Protein ◽

Aryl Hydrocarbon

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a photoreceptor specific chaperone of the visual effector enzyme phosphodiesterase-6 (PDE6). AIPL1 has been shown to bind the farnesylated PDE6A subunit. Mutations in AIPL1 are thought to destabilize PDE6 and thereby cause Leber congenital amaurosis type 4 (LCA4), a severe form of childhood blindness. Here, we examined the solution structure of AIPL1 by small angle x-ray scattering. A structural model of AIPL1 with the best fit to the scattering data features two independent FK506-binding protein (FKBP)-like and tetratricopeptide repeat domains. Guided by the model, we tested the hypothesis that AIPL1 directly binds the farnesyl moiety. Our studies revealed high affinity binding of the farnesylated-Cys probe to the FKBP-like domain of AIPL1, thus uncovering a novel function of this domain. Mutational analysis of the potential farnesyl-binding sites on AIPL1 identified two critical residues, Cys-89 and Leu-147, located in close proximity in the structure model. The L147A mutation and the LCA-linked C89R mutation prevented the binding of the farnesyl-Cys probe to AIPL1. Furthermore, Cys-89 and Leu-147 flank the unique insert region of AIPL1, deletion of which also abolished the farnesyl interaction. Our results suggest that the binding of PDE6A farnesyl is essential to normal function of AIPL1 and its disruption is one of the mechanisms underlying LCA.

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Multivalent assembly of KRAS with the RAS-binding and cysteine-rich domains of CRAF on the membrane

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914076117 ◽

2020 ◽

Vol 117 (22) ◽

pp. 12101-12108 ◽

Cited By ~ 6

Author(s):

Zhenhao Fang ◽

Ki-Young Lee ◽

Ku-Geng Huo ◽

Geneviève Gasmi-Seabrook ◽

Le Zheng ◽

...

Keyword(s):

Electrostatic Interactions ◽

Kinase Domain ◽

Mapk Signaling ◽

Lipid Binding ◽

Nmr Studies ◽

Dynamic Interactions ◽

Rich Domain ◽

C Terminus ◽

Membrane Interfaces ◽

Dynamics Simulations

Membrane anchoring of farnesylated KRAS is critical for activation of RAF kinases, yet our understanding of how these proteins interact on the membrane is limited to isolated domains. The RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF engage KRAS and the plasma membrane, unleashing the kinase domain from autoinhibition. Due to experimental challenges, structural insight into this tripartite KRAS:RBD–CRD:membrane complex has relied on molecular dynamics simulations. Here, we report NMR studies of the KRAS:CRAF RBD–CRD complex. We found that the nucleotide-dependent KRAS–RBD interaction results in transient electrostatic interactions between KRAS and CRD, and we mapped the membrane interfaces of the CRD, RBD–CRD, and the KRAS:RBD–CRD complex. RBD–CRD exhibits dynamic interactions with the membrane through the canonical CRD lipid-binding site (CRD β7–8), as well as an alternative interface comprising β6 and the C terminus of CRD and β2 of RBD. Upon complex formation with KRAS, two distinct states were observed by NMR: State A was stabilized by membrane association of CRD β7–8 and KRAS α4–α5 while state B involved the C terminus of CRD, β3–5 of RBD, and part of KRAS α5. Notably, α4–α5, which has been proposed to mediate KRAS dimerization, is accessible only in state B. A cancer-associated mutation on the state B membrane interface of CRAF RBD (E125K) stabilized state B and enhanced kinase activity and cellular MAPK signaling. These studies revealed a dynamic picture of the assembly of the KRAS–CRAF complex via multivalent and dynamic interactions between KRAS, CRAF RBD–CRD, and the membrane.

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