2,4-Dinitrophenol reduces the reactivity of Lys553 in the lower 50-kDa region of myosin subfragment 1

• Published in: Archives of biochemistry and biophysics 2011, Vol.505 (1), p.105-111
• Main Authors: Bomfim, Theresa R., Machado, Luciana E.S.F., Lima, Luis Mauricio T.R., Sorenson, Martha M., Salerno, Verônica P.
• Format: Article
• Language: English
• Subjects: 2,4-Dinitrophenol - pharmacology; Actin; Actins - metabolism; Adenosine Triphosphate - metabolism; Animals; Binding Sites; Coloring Agents - pharmacology; DNP; FHS; Fluorescence; Fluorescent Dyes; Helix-Loop-Helix Motifs; Lysine - metabolism; Myosin Subfragments - chemistry; Myosin Subfragments - metabolism; Protein Binding; Protein Conformation - drug effects; Rabbits; Subfragment 1
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2010.09.022

► The compound 2,4-dinitrophenol (DNP) binds near a helix-loop-helix that includes a reactive lysine (Lys553) in myosin’s lower 50-kDa subdomain, activating ATP hydrolysis. ► Reactivity of this lysine is reduced differentially by a broad range of ligands (nucleotide analogs, actin and DNP), indicating significant conformational flexibility among subdomains along the entire pathway from ATP pocket to the actin-binding region. ► Lys553 reactivity appears to be correlated with progressive closure of the 50-kDa cleft rather than changes in the status of switch 2. We suggest the DNP stabilizes the cleft in a partially open state. 2,4-Dinitrophenol (DNP) increases the affinity of myosin for actin and accelerates its Mg 2+ATPase activity, suggesting that it acts on a region of the myosin head that transmits conformational changes to actin- and ATP-binding sites. The binding site/s for DNP are unknown; however similar hydrophobic compounds bind to the 50-kDa subfragment of the myosin head, near the actin-binding interface. In this region, a helix-loop-helix motif contains Lys553, which is specifically labeled with the fluorescent probe 6-[fluorescein-5(and 6)-carboxamido] hexanoic acid succinimidyl ester (FHS). This reaction is sensitive to conformational changes in the helix-loop-helix and the labeling efficiency was reduced when S1 was bound to actin, DNP or nucleotide analogs. The nucleotide analogs had a range of effects (PPi > ADP·AlF 4 − > ADP) irrespective of the open-closed state of switch 2. The greatest reduction in labeling was in the presence of actin or DNP. When we measured the effect of each ligand on the fluorescence of FHS previously attached to S1, only DNP quenched the emission. Together, the results suggest that the helix-loop-helix region is flexible, it is part of the communication pathway between the ATP- and actin-binding sites of myosin and it is proximal to the region of myosin where DNP binds.