title: Expression and Purification of ZASP Subdomains and Clinically Important Isoforms: High-Affinity Binding to G-Actin.
abstract: Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP) is a principal component of the sarcomere. The three prevalent isoforms of ZASP in skeletal muscle are generated by alternative splicing of exons 9 and 10. The long isoforms, either having (ZASP-L) or lacking exon 10 (ZASP-LΔex10), include an N-terminal PDZ domain, an actin-binding region (ABR) with a conserved motif (ZM), and three C-terminal LIM domains. The short isoform (ZASP-S) lacks the LIM domains. Mutations, A147T and A165V, within the ZM of ZASP-LΔex10 cause myofibrillar myopathy, but the mechanism is unknown. We have prepared these proteins, their ABR, and the respective mutant variants in recombinant form, characterized them biophysically, and analyzed their actin-binding properties by surface plasmon resonance and electron microscopy. All the proteins were physically homogeneous and monomeric and had circular dichroic spectra consistent with partially folded conformations. Comparison of the NMR HSQC spectra of ZASP-S and the PDZ domain showed that the ABR is unstructured. ZASP-S and its mutant variants and ZASP-LΔex10 all bound to immobilized G-actin with high affinity (Kd≈ 10-8to 10-9M). Constructs of the isolated actin-binding region missing exon 10 (ABRΔ10) bound with lower affinity (Kd≈ 10-7M), but those retaining exon 10 (ABR+10) did so only weakly (Kd≈ 10-5M). ZASP-S, and the ABRΔ10, also induced F-actin and array formation, even in conditions of low ionic strength and in the absence of KCl and Mg2+ions. Interestingly, the ZM mutations A147T and A165V did not affect any of the results described above.
authors.forename: Norman R
authors.initials: NR
authors.lastname: Watts
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Zhuang
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Kaufman
authors.forename: Ira W
authors.initials: IW
authors.lastname: Palmer
authors.forename: Altaira D
authors.initials: AD
authors.lastname: Dearborn
authors.forename: Stephen
authors.initials: S
authors.lastname: Coscia
authors.forename: Yotam
authors.initials: Y
authors.lastname: Blech-Hermoni
authors.affiliation: Department of Clinical and Basic Neuroscience, King's College London , London SE5 8AF, United Kingdom.
authors.forename: Caterina
authors.initials: C
authors.lastname: Alfano
authors.affiliation: Department of Clinical and Basic Neuroscience, King's College London , London SE5 8AF, United Kingdom.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Pastore
authors.forename: Ami
authors.initials: A
authors.lastname: Mankodi
authors.forename: Paul T
authors.identifier: 0000-0002-9515-4752
authors.initials: PT
authors.lastname: Wingfield
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017398:Alternative Splicing; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005091:Exons; D015870:Gene Expression; D006801:Humans; D007438:Introns; D060588:LIM Domain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009994:Osmolar Concentration; D011485:Protein Binding; D000072417:Protein Domains; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012518:Sarcomeres / Q000502:physiology; D013329:Structure-Activity Relationship
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C120696:LDB3 protein, human; D060588:LIM Domain Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins