F-actin structure destabilization and DNase I binding loop: fluctuations mutational cross-linking and electron microscopy analysis of loop states and effects on F-actin.
The conformational dynamics of filamentous actin (F-actin) is essential for the regulation and functions of cellular actin networks. The main contribution to F-actin dynamics and its multiple conformational states arises from the mobility and flexibility of the DNase I binding loop (D-loop; residues 40-50) on subdomain 2. Therefore, we explored the structural constraints on D-loop plasticity at the F-actin interprotomer space by probing its dynamic interactions with the hydrophobic loop (H-loop), the C-terminus, and the W-loop via mutational disulfide cross-linking. To this end, residues of the D-loop were mutated to cysteines on yeast actin with a C374A background. These mutants showed no major changes in their polymerization and nucleotide exchange properties compared to wild-type actin. Copper-catalyzed disulfide cross-linking was investigated in equimolar copolymers of cysteine mutants from the D-loop with either wild-type (C374) actin or mutant S265C/C374A (on the H-loop) or mutant F169C/C374A (on the W-loop). Remarkably, all tested residues of the D-loop could be cross-linked to residues 374, 265, and 169 by disulfide bonds, demonstrating the plasticity of the interprotomer region. However, each cross-link resulted in different effects on the filament structure, as detected by electron microscopy and light-scattering measurements. Disulfide cross-linking in the longitudinal orientation produced mostly no visible changes in filament morphology, whereas the cross-linking of D-loop residues >45 to the H-loop, in the lateral direction, resulted in filament disruption and the presence of amorphous aggregates on electron microscopy images. A similar aggregation was also observed upon cross-linking the residues of the D-loop (>41) to residue 169. The effects of disulfide cross-links on F-actin stability were only partially accounted for by the simulations of current F-actin models. Thus, our results present evidence for the high level of conformational plasticity in the interprotomer space and document the link between D-loop interactions and F-actin stability.
Oztug Durer
Zeynep A
ZA
Department of Chemistry and Biochemistry, and Molecular Biology Institute, University of California at Los Angeles, CA 90095, USA.
Diraviyam
Karthikeyan
K
Sept
David
D
Kudryashov
Dmitri S
DS
Reisler
Emil
E
D000199:Actins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D019943:Amino Acid Substitution; D001483:Base Sequence; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D003850:Deoxyribonuclease I / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D055503:Protein Multimerization; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D013816:Thermodynamics
D000199:Actins; D003432:Cross-Linking Reagents; D017931:DNA Primers; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D003850:Deoxyribonuclease I; D003545:Cysteine