Structural basis of the filamin A actin-binding domain interaction with F-actin

Cryo-EM reconstructions were deposited in the Electron Microscopy Data Bank with the following accession numbers: F20-F-actin-FLNaABD, EMD-7833; F20-F-actin-FLNaABD-Q170P, EMD-7832; F20-F-actin-FLNaABD-E254K, EMD-8918; Krios-F-actin-FLNaABD-E254K, EMD-7831. The corresponding FLNaABD-E254K filament model was deposited in the PDB with accession number 6D8C. Source data for F-actin-targeting analyses (Figs. 2c,d,g,h, 3b,c,e,f, 4d,e, 5c,d, and 6a,b) and co-sedimentation assays (Figs. 5g and 6d) are available with the paper online. Other data are available from the corresponding author upon reasonable request. We thank Z. Razinia for generating numerous FLNa constructs, S. Wu for expertise in using the Krios microscope, J. Lees for advice on model refinement, and M. Lemmon for helpful comments in preparing the manuscript. We also thank the Yale Center for Research Computing for guidance and use of the Farnam Cluster, as well as the staff at the YMS Center for Molecular Imaging for the use of the EM Core Facility. This work was funded by grants from the National Institutes of Health (R01-GM068600 (D.A.C.), R01-NS093704 (D.A.C.), R37-GM057247 (C.V.S.), R01-GM110530 (C.V.S.), T32-GM007324, T32-GM008283) and an award from American Heart Association (15PRE25700119 (D.V.I.)).Peer reviewedPostprin

Assembly of a filamin four-domain fragment and the influence of splicing variant-1 on the structure.

Filamins are scaffold proteins that bind to various proteins, including the actin cytoskeleton, integrin adhesion receptors, and adaptor proteins such as migfilin. Alternative splicing of filamin, largely constructed from 24 Ig-like domains, is thought to have a role in regulating its interactions with other proteins. The filamin A splice variant-1 (FLNa var-1) lacks 41 amino acids, including the last β-strand of domain 19, FLNa(19), and the first β-strand of FLNa(20) that was previously shown to mask a key binding site on FLNa(21). Here, we present a structural characterization of domains 18-21, FLNa(18-21), in the FLNa var-1 as well as its nonspliced counterpart. A model of nonspliced FLNa(18-21), obtained from small angle x-ray scattering data, shows that these four domains form an L-shaped structure, with one arm composed of a pair of domains. NMR spectroscopy reveals that in the splice variant, FLNa(19) is unstructured whereas the other domains retain the same fold as in their canonical counterparts. The maximum dimensions predicted by small angle x-ray scattering data are increased upon migfilin binding in the FLNa(18-21) but not in the splice variant, suggesting that migfilin binding is able to displace the masking β-strand and cause a rearrangement of the structure. Possible function roles for the spliced variants are discussed

Assembly of a filamin four-domain fragment and the influence of splicing variant-1 on the structure.

Filamins are scaffold proteins that bind to various proteins, including the actin cytoskeleton, integrin adhesion receptors, and adaptor proteins such as migfilin. Alternative splicing of filamin, largely constructed from 24 Ig-like domains, is thought to have a role in regulating its interactions with other proteins. The filamin A splice variant-1 (FLNa var-1) lacks 41 amino acids, including the last β-strand of domain 19, FLNa(19), and the first β-strand of FLNa(20) that was previously shown to mask a key binding site on FLNa(21). Here, we present a structural characterization of domains 18-21, FLNa(18-21), in the FLNa var-1 as well as its nonspliced counterpart. A model of nonspliced FLNa(18-21), obtained from small angle x-ray scattering data, shows that these four domains form an L-shaped structure, with one arm composed of a pair of domains. NMR spectroscopy reveals that in the splice variant, FLNa(19) is unstructured whereas the other domains retain the same fold as in their canonical counterparts. The maximum dimensions predicted by small angle x-ray scattering data are increased upon migfilin binding in the FLNa(18-21) but not in the splice variant, suggesting that migfilin binding is able to displace the masking β-strand and cause a rearrangement of the structure. Possible function roles for the spliced variants are discussed

Juxtanodin is an intrinsically disordered F-actin-binding protein

Juxtanodin, also called ermin, is an F-actin-binding protein expressed by oligodendrocytes, the myelin-forming cells of the central nervous system. While juxtanodin carries a short conserved F-actin-binding segment at its C terminus, it otherwise shares no similarity with known protein sequences. We carried out a structural characterization of recombinant juxtanodin in solution. Juxtanodin turned out to be intrinsically disordered, as evidenced by conventional and synchrotron radiation CD spectroscopy. Small-angle X-ray scattering indicated that juxtanodin is a monomeric, highly elongated, unfolded molecule. Ensemble optimization analysis of the data suggested also the presence of more compact forms of juxtanodin. The C terminus was a strict requirement for co-sedimentation of juxtanodin with microfilaments, but juxtanodin had only mild effects on actin polymerization. The disordered nature of juxtanodin may predict functions as a protein interaction hub, although F-actin is its only currently known binding partner

The intracellular domain of BP180/collagen XVII is intrinsically disordered and partially folds in an anionic membrane lipid-mimicking environment

Abstract The trimeric transmembrane collagen BP180, also known as collagen XVII, is an essential component of hemidesmosomes at the dermal–epidermal junction and connects the cytoplasmic keratin network to the extracellular basement membrane. Dysfunction of BP180 caused by mutations in patients with junctional epidermolysis bullosa or autoantibodies in those with bullous pemphigoid leads to severe skin blistering. The extracellular collagenous domain of BP180 participates in the protein’s triple-helical folding, but the structure and functional importance of the intracellular domain (ICD) of BP180 are largely unknown. In the present study, we purified and characterized human BP180 ICD. When expressed in Escherichia coli as glutathione-S-transferase or 6 × histidine tagged fusion protein, the BP180 ICD was found to exist as a monomer. Analysis of the secondary structure content by circular dichroism spectroscopy revealed that the domain is intrinsically disordered. This finding aligned with that of a bioinformatic analysis, which predicted a disordered structure. Interestingly, both anionic detergent micelles and lipid vesicles induced partial folding of the BP180 ICD, suggesting that in its natural environment, the domain’s folding and unfolding may be regulated by interaction with the cell membrane or accompanying proteins. We hypothesize that the intrinsically disordered structure of the ICD of BP180 contributes to the mechanism that allows the remodeling of hemidesmosome assembly

The C-terminal rod 2 fragment of filamin A forms a compact structure that can be extended

Filamins are large proteins that cross-link actin filaments and connect to other cellular components. The C-terminal rod 2 region of FLNa (filamin A) mediates dimerization and interacts with several transmembrane receptors and intracellular signalling adaptors. SAXS (small-angle X-ray scattering) experiments were used to make a model of a six immunoglobulin-like domain fragment of the FLNa rod 2 (domains 16-21). This fragment had a surprising three-branched structural arrangement, where each branch was made of a tightly packed two-domain pair. Peptides derived from transmembrane receptors and intracellular signalling proteins induced a more open structure of the six domain fragment. Mutagenesis studies suggested that these changes are caused by peptides binding to the CD faces on domains 19 and 21 which displace the preceding domain A-strands (18 and 20 respectively), thus opening the individual domain pairs. A single particle cryo-EM map of a nine domain rod 2 fragment (domains 16-24), showed a relatively compact dimeric particle and confirmed the three-branched arrangement as well as the peptide-induced conformation changes. These findings reveal features of filamin structure that are important for its interactions and mechanical properties

Conformational analysis of membrane-proximal segments of GDAP1 in a lipidic environment using synchrotron radiation suggests a mode of assembly at the mitochondrial outer membrane

Abstract The mitochondrial outer membrane creates a diffusion barrier between the cytosol and the mitochondrial intermembrane space, allowing the exchange of metabolic products, important for efficient mitochondrial function in neurons. The ganglioside-induced differentiation-associated protein 1 (GDAP1) is a mitochondrial outer membrane protein with a critical role in mitochondrial dynamics and metabolic balance in neurons. Missense mutations in the GDAP1 gene are linked to the most common human peripheral neuropathy, Charcot-Marie-Tooth disease (CMT). GDAP1 is a distant member of the glutathione-S-transferase (GST) superfamily, with unknown enzymatic properties or functions at the molecular level. The structure of the cytosol-facing GST-like domain has been described, but there is no consensus on how the protein interacts with the mitochondrial outer membrane. Here, we describe a model for GDAP1 assembly on the membrane using peptides vicinal to the GDAP1 transmembrane domain. We used oriented circular dichroism spectroscopy (OCD) with synchrotron radiation to study the secondary structure and orientation of GDAP1 segments at the outer and inner surfaces of the outer mitochondrial membrane. These experiments were complemented by small-angle X-ray scattering, providing the first experimental structural models for full-length human GDAP1. The results indicate that GDAP1 is bound into the membrane via a single transmembrane helix, flanked by two peripheral helices interacting with the outer and inner leaflets of the mitochondrial outer membrane in different orientations. Impairment of these interactions could be a mechanism for CMT in the case of missense mutations affecting these segments instead of the GST-like domain

Conserved intramolecular networks in GDAP1 are closely connected to CMT-linked mutations and protein stability

Abstract Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral polyneuropathy in humans, and its subtypes are linked to mutations in dozens of different genes, including the gene coding for ganglioside-induced differentiation-associated protein 1 (GDAP1). The main GDAP1-linked CMT subtypes are the demyelinating CMT4A and the axonal CMT2K. Over a hundred different missense CMT mutations in the GDAP1 gene have been reported. However, despite implications for mitochondrial fission and fusion, cytoskeletal interactions, and response to reactive oxygen species, the etiology of GDAP1-linked CMT is poorly understood at the protein level. Based on earlier structural data, CMT-linked mutations could affect intramolecular interaction networks within the GDAP1 protein. We carried out structural and biophysical analyses on several CMT-linked GDAP1 protein variants and describe new crystal structures of the autosomal recessive R120Q and the autosomal dominant A247V and R282H GDAP1 variants. These mutations reside in the structurally central helices ⍺3, ⍺7, and ⍺8. In addition, solution properties of the CMT mutants R161H, H256R, R310Q, and R310W were analysed. All disease variant proteins retain close to normal structure and solution behaviour. All mutations, apart from those affecting Arg310 outside the folded GDAP1 core domain, decreased thermal stability. In addition, a bioinformatics analysis was carried out to shed light on the conservation and evolution of GDAP1, which is an outlier member of the GST superfamily. GDAP1-like proteins branched early from the larger group of GSTs. Phylogenetic calculations could not resolve the exact early chronology, but the evolution of GDAP1 is roughly as old as the splits of archaea from other kingdoms. Many known CMT mutation sites involve conserved residues or interact with them. A central role for the ⍺6-⍺7 loop, within a conserved interaction network, is identified for GDAP1 protein stability. To conclude, we have expanded the structural analysis on GDAP1, strengthening the hypothesis that alterations in conserved intramolecular interactions may alter GDAP1 stability and function, eventually leading to mitochondrial dysfunction, impaired protein-protein interactions, and neuronal degeneration