Expression and Purification of ZASP Subdomains and Clinically Important Isoforms: High-Affinity Binding to G-Actin.

Norman R Watts, Xiaolei Zhuang, Joshua D Kaufman, Ira W Palmer, Altaira D Dearborn, Stephen Coscia, Yotam Blech-Hermoni, Caterina Alfano, Annalisa Pastore, Ami Mankodi, Paul T Wingfield

Biochemistry 2017 Apr 11

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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 ≈ 10-8 to 10-9 M). Constructs of the isolated actin-binding region missing exon 10 (ABRΔ10) bound with lower affinity (Kd ≈ 10-7 M), but those retaining exon 10 (ABR+10) did so only weakly (Kd ≈ 10-5 M). 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.

Citation

Norman R Watts, Xiaolei Zhuang, Joshua D Kaufman, Ira W Palmer, Altaira D Dearborn, Stephen Coscia, Yotam Blech-Hermoni, Caterina Alfano, Annalisa Pastore, Ami Mankodi, Paul T Wingfield. Expression and Purification of ZASP Subdomains and Clinically Important Isoforms: High-Affinity Binding to G-Actin. Biochemistry. 2017 Apr 11;56(14):2061-2070