Structural basis of Fusarium myosin I inhibition by phenamacril

Abstract

Fusarium is a genus of filamentous fungi that includes species that cause devastating diseases in major staple crops, such as wheat, maize, rice, and barley, resulting in severe yield losses and mycotoxin contamination of infected grains. Phenamacril is a novel fungicide that is considered environmentally benign due to its exceptional specificity; it inhibits the ATPase activity of the sole class I myosin of only a subset of Fusarium species including the major plant pathogens F. graminearum, F. asiaticum and F. fujikuroi. To understand the underlying mechanisms of inhibition, species specificity, and resistance mutations, we have determined the crystal structure of phenamacril-bound F. graminearum myosin I. Phenamacril binds in the actin-binding cleft in a new allosteric pocket that contains the central residue of the regulatory Switch 2 loop and that is collapsed in the structure of a myosin with closed actin-binding cleft, suggesting that pocket occupancy blocks cleft closure. We have further identified a single, transferable phenamacril-binding residue found exclusively in phenamacril-sensitive myosins to confer phenamacril selectivity.

Structural and Computational Insights into a Blebbistatin-Bound Myosin•ADP Complex with Characteristics of an ADP-Release Conformation along the Two-Step Myosin Power Stoke

“…In addition, our simulations indicate that myosin alone, in the absence of the inhibitor blebbistatin, can adopt a conformation during the second converter rotation that shows high similarities with the myosin conformation in our crystallized myosin-II·ADP·blebbistatin complex, with all critical structural elements, involved in chemomechanical coupling, in comparable positions. This is in good agreement with various earlier studies, which showed that other known myosin modulators trap the myosin motor in distinct conformational states of the motor cycle, inducing only smaller local structural changes [ 14 , 15 , 16 , 17 ]. The applied simulations were performed in the absence of blebbistatin and were unbiased in the way that the myosin-II·ADP·blebbistatin crystal structure was not included during our TMD simulations, but we induced the power stroke by forcing the distant converter to rotate, which eventually led to associated changes in the motor domain and particularly the active site, thereby populating a conformation that resembled our blebbistatin-bound myosin-II structure.…”

“…Computational and energetic reconstruction of the ADP release pathway from the crystallographically resolved pre-power stroke state (with a strongly bound ADP) up to nucleotide dissociation from the motor domain suggests the population of low-energy conformations along the release pathway that highly resemble both the proposed strong-Mg 2+ ·ADP-bound state and the myosin conformation as seen in our blebbistatin-bound myosin-II·ADP crystal structure, without including the two experimental structures in the computations a priori. Hence, myosin alone seems to be able to adopt a similar conformation as our crystallized inhibitor-bound myosin structure, which is in good agreement with studies on various small molecule myosin inhibitors [ 14 , 15 , 17 ] and activators [ 16 ] that trap myosin in specific conformations that can be assigned as structural states of the actomyosin motor cycle. These results further corroborate the hypothesis that the overall crystallized structure in the presence of blebbistatin resembles a native myosin-II conformation along the power stroke.…”

“…In conclusion, the overall conformation and particularly the positions of the characteristic structural elements involved in chemomechanical coupling suggest that our crystallized myosin-II·ADP·blebbistatin structure resembles a myosin conformation towards the end of the power stroke, involved in the second step of converter/lever arm swing during ADP release, following the strong-Mg 2+ ·ADP-bound myosin state. Although we cannot fully exclude on the basis of our data the possibility that the conformation in our crystal structure is only a consequence of the bound inhibitor blebbistatin, and the ADP-release conformation of myosin-II is significantly different, previous results with other small molecule myosin modulators [ 14 , 15 , 16 , 17 ], which trapped myosin in distinct states along the actomyosin motor cycle, and specifically the positions of the critical structural elements of the motor strongly foster our hypothesis that the crystallized myosin-II structure complexed with ADP and blebbistatin is very similar to the native myosin ADP-release conformation. Considering that the structure represents the ADP-release conformation, we postulate that the transition from the strong-Mg 2+ ·ADP-bound myosin state as observed with cryoEM [ 12 , 13 , 31 ] to the ADP-release conformation might be driven by the release of Mg 2+ , which leads to repositioning of the switches in the active site, coupled to the initiation of the second converter/lever arm rotation.…”

“…The myosin motor is thus subject to substantial conformational changes along that cycle, traversing through different actin-attached and –detached states, eventually amplifying them to a large scale rotation of the converter domain and swinging of the adjacent lever arm in the force-generating power stroke [ 10 ]. Many of the subprocesses have been extensively studied in the past, and newly observed conformations, obtained through cryo-electron microscopy (cryoEM) [ 11 , 12 , 13 ] or crystallization with native and artificial ligands [ 14 , 15 , 16 , 17 ] that trapped myosin in a specific state, have been assigned as states of the actomyosin cycle according to the status of the characteristic structural elements in the myosin motor domain, thus building the basis for the motor function. Numerous studies have significantly contributed to our understanding of the mechanism of the recovery stroke [ 18 , 19 , 20 , 21 , 22 , 23 ], which primes the myosin motor for force production while dissociated from the actin filament.…”

Review on Structures of Pesticide Targets

“…Myosin I is an important target in Fusarium graminearum [ 33 ], and the complex crystal structure of phenamacril-bound myosin I in F. graminearum was solved. It was discovered that phenamacril binds in the actin-binding cleft of a new allosteric pocket ( Figure 2 C) [ 34 ].…”