bi5b00082_si_001.pdf (1.46 MB)
Dissection of the Water Cavity of Yeast Thioredoxin 1: The Effect of a Hydrophobic Residue in the Cavity
journal contribution
posted on 2015-04-21, 00:00 authored by Anwar Iqbal, Francisco Gomes-Neto, Catarina
Akiko Myiamoto, Ana Paula Valente, Fabio C. L. AlmeidaThe
water cavity of yeast thioredoxin 1 (yTrx1) is an ancestral,
conserved structural element that is poorly understood. We recently
demonstrated that the water cavity is involved in the complex protein
dynamics that are responsible for the catalytically relevant event
of coupling hydration, proton exchange, and motion at the interacting
loops. Its main feature is the presence of the conserved polar residue,
Asp24, which is buried in a hydrophobic cavity. Here, we evaluated
the role of the solvation of Asp24 as the main element that is responsible
for the formation of the water cavity in thioredoxins. We showed that
the substitution of Asp24 with a hydrophobic residue (D24A) was not
sufficient to completely close the cavity. The dynamics of the D24A
mutant of yTrx1 at multiple time scales revealed that the D24A mutant
presents motions at different time scales near the active site, interaction
loops, and water cavity, revealing the existence of a smaller dissected
cavity. Molecular dynamics simulation, along with experimental molecular
dynamics, allowed a detailed description of the water cavity in wild-type
yTrx1 and D24A. The cavity connects the interacting loops, the central
β-sheet, and α-helices 2 and 4. It is formed by three
contiguous lobes, which we call lobes A–C. Lobe A is hydrophilic
and the most superficial. It is formed primarily by the conserved
Lys54. Lobe B is the central lobe formed by the catalytically important
residues Cys33 and Asp24, which are strategically positioned. Lobe
C is the most hydrophobic and is formed by the conserved cis-Pro73. The central lobe B is closed upon introduction of the D24A
mutation, revealing that independent forces other than solvation of
Asp24 maintain lobes A and C in the open configuration. These data
allow us to better understand the properties of this enzyme.