2WA6

Structure of the W148R mutant of human filamin b actin binding domain at 1.95 Angstrom resolution

Summary for 2WA6

• Entry DOI: 10.2210/pdb2wa6/pdb
• Related: 2DI8 2DI9 2DIA 2DIB 2DIC 2DJ4 2WA5 2WA7
• Descriptor: FILAMIN-B, CARBONATE ION (3 entities in total)
• Functional Keywords: disease mutation, skeletal dysplasia, structural protein, actin-crosslinking, myogenesis, cytoskeleton, actin-binding, calponin homology domain, phosphoprotein, differentiation, atelosteogenesis, mutant, filamin, dwarfism, ch domain, developmental protein
• Biological source: HOMO SAPIENS (HUMAN)
• Cellular location: Isoform 1: Cytoplasm, cell cortex. Isoform 2: Cytoplasm, cytoskeleton. Isoform 3: Cytoplasm, cytoskeleton. Isoform 6: Cytoplasm, cytoskeleton: O75369
• Total number of polymer chains: 1
• Total formula weight: 28226.19

Authors

Sawyer, G.M.,Clark, A.R.,Robertson, S.P.,Sutherland-Smith, A.J. (deposition date: 2009-02-03, release date: 2009-06-23, Last modification date: 2023-12-13)

Primary citation

Sawyer, G.M.,Clark, A.R.,Robertson, S.P.,Sutherland-Smith, A.J.
Disease-Associated Substitutions in the Filamin B Actin Binding Domain Confer Enhanced Actin Binding Affinity in the Absence of Major Structural Disturbance: Insights from the Crystal Structures of Filamin B Actin Binding Domains.
J.Mol.Biol., 390:1030-, 2009

PubMed Abstract: Missense mutations in filamin B (FLNB) are associated with the autosomal dominant atelosteogenesis (AO) and the Larsen group of skeletal malformation disorders. These mutations cluster in particular FLNB protein domains and act in a presumptive gain-of-function mechanism. In contrast the loss-of-function disorder, spondylocarpotarsal synostosis syndrome, is characterised by the complete absence of FLNB. One cluster of AO missense mutations is found within the second of two calponin homology (CH) domains that create a functional actin-binding domain (ABD). This N-terminal ABD is required for filamin F-actin crosslinking activity, a crucial aspect of filamin's role of integrating cell-signalling events with cellular scaffolding and mechanoprotection. This study characterises the wild type FLNB ABD and investigates the effects of two disease-associated mutations on the structure and function of the FLNB ABD that could explain a gain-of-function mechanism for the AO diseases. We have determined high-resolution X-ray crystal structures of the human filamin B wild type ABD, plus W148R and M202V mutants. All three structures display the classic compact monomeric conformation for the ABD with the CH1 and CH2 domains in close contact. The conservation of tertiary structure in the presence of these mutations shows that the compact ABD conformation is stable to the sequence substitutions. In solution the mutant ABDs display reduced melting temperatures (by 6-7 degrees C) as determined by differential scanning fluorimetry. Characterisation of the wild type and mutant ABD F-actin binding activities via co-sedimentation assays shows that the mutant FLNB ABDs have increased F-actin binding affinities, with dissociation constants of 2.0 microM (W148R) and 0.56 microM (M202V), compared to the wild type ABD K(d) of 7.0 microM. The increased F-actin binding affinity of the mutants presents a biochemical mechanism that differentiates the autosomal dominant gain-of-function FLNB disorders from those that arise through the complete loss of FLNB protein.

PubMed: 19505475
DOI: 10.1016/J.JMB.2009.06.009

Experimental method

X-RAY DIFFRACTION (1.95 Å)