Dematin exhibits a natively unfolded core domain and an independently folded headpiece domain.
Dematin is an actin-binding protein originally identified in the junctional complex of the erythrocyte plasma membrane, and is present in many nonerythroid cells. Dematin headpiece knockout mice display a spherical red cell phenotype and develop a compensated anemia. Dematin has two domains: a 315-residue, proline-rich "core" domain and a 68-residue carboxyl-terminal villin-type "headpiece" domain. Expression of full-length dematin in E. coli as a GST recombinant protein results in truncation within a proline, glutamic acid, serine, threonine rich region (PEST). Therefore, we designed a mutant construct that replaces the PEST sequence. The modified dematin has high actin binding activity as determined by actin sedimentation assays. Negative stain electron microscopy demonstrates that the modified dematin also exhibits actin bundling activity like that of native dematin. Circular dichroism (CD) and NMR spectral analysis, however, show little secondary structure in the modified dematin. The lack of secondary structure is also observed in native dematin purified from human red blood cells. (15)N-HSQC NMR spectra of modified dematin indicate that the headpiece domain is fully folded whereas the core region is primarily unfolded. Our finding suggests that the core is natively unfolded and may serve as a scaffold to organize the components of the junctional complex.
Chen
Lin
L
Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Jiang
Zhenghui G
ZG
Khan
Anwar A
AA
Chishti
Athar H
AH
McKnight
C James
CJ
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance
D000199:Actins; D001798:Blood Proteins; C044805:DMTN protein, human; D008840:Microfilament Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins