Understanding the apparent stator‐rotor connections in the rotary ATPase family using coarse‐grained computer modeling

Abstract

Advances in structural biology, such as cryo-electron microscopy (cryo-EM) have allowed for a number of sophisticated protein complexes to be characterized. However, often only a static snapshot of a protein complex is visualized despite the fact that conformational change is frequently inherent to biological function, as is the case for molecular motors. Computer simulations provide valuable insights into the different conformations available to a particular system that are not accessible using conventional structural techniques. For larger proteins and protein complexes, where a fully atomistic description would be computationally prohibitive, coarse-grained simulation techniques such as Elastic Network Modeling (ENM) are often employed, whereby each atom or group of atoms is linked by a set of springs whose properties can be customized according to the system of interest. Here we compare ENM with a recently proposed continuum model known as Fluctuating Finite Element Analysis (FFEA), which represents the biomolecule as a viscoelastic solid subject to thermal fluctuations. These two complementary computational techniques are used to answer a critical question in the rotary ATPase family; implicit within these motors is the need for a rotor axle and proton pump to rotate freely of the motor domain and stator structures. However, current single particle cryo-EM reconstructions have shown an apparent connection between the stators and rotor axle or pump region, hindering rotation. Both modeling approaches show a possible role for this connection and how it would significantly constrain the mobility of the rotary ATPase family.

Methods to account for movement and flexibility in cryo-EM data processing

“…For all of these studies, inherent flexibility has been overcome through the use of masking out specific regions of interest. For this study we have used the V-ATPase as a model system as it works through a rotary mechanism which involves structural flexibility as seen in 2-D image classes from electron microscopy (EM) [21] , and molecular dynamics simulations [22] , [23] . Compared to a series of discrete states, the continuous nature of this flexing makes sorting of the heterogeneity difficult by conventional 3-D classification.…”

Structure of the Vacuolar H + -ATPase Rotary Motor Reveals New Mechanistic Insights

“…Constraint by the bearing could also contribute to keeping the foot of the D subunit engaged with subunit d , which couples the axle to the c ring. D- d coupling is predicted by fluctuating finite element analysis modeling to be relatively loose ( Richardson et al., 2014 ) and may play a role in the structural rearrangements associated with ATP silencing in the absence of glucose ( Sumner et al., 1995; Kane, 1995; Tabke et al., 2014 ). F-ATPase shows a similar charged feature in the motor domain with highly conserved PGREAYP (α subunit) and PSAVGYQP (β subunit) motifs ( Table 2 ).…”

“…A conserved feature in all published A/V-ATPase reconstructions is a density that apparently connects the final stator (S3 in V-ATPase and S2 in A-ATPase) to subunit d ( Figure 4 C). Anomalously, this would hinder the free rotation of the axle relative to the stator network, which in molecular dynamics (MD) simulations increases complex stability ( Richardson et al., 2014 ). At ∼1 nm resolution this feature is weaker, indicative of a close but crucially noncontacting surface ( Figure 4 D).…”

In pursuit of an accurate spatial and temporal model of biomolecules at the atomistic level: a perspective on computer simulation

“…These material parameters should describe the cumulative effect at the continuum level of all of the local atomic interactions. Once these parameters have been defined, the trajectory describing the changing shape of the protein owing to thermal fluctuations can be calculated by iteratively integrating Newton's equations of motion over short timesteps and moving each node of the mesh accordingly, as shown for the rotary ATPase motor in Supplementary Movie S6 (Richardson et al, 2014). The calculation is analogous to conventional molecular dynamics (MD), but the forces on each node within the mesh are derived from continuum mechanics equations rather than a pairwise particle-based force field.…”

“…The value of these models lies in their simplicity: to calculate the normal modes of a protein of interest a user simply needs to upload the PDB file (for example to the elNémo webserver; Suhre & Sanejouand, 2004) and download the results. From left to right, this figure shows the EMDB density map of the Thermus thermophilus A-ATPase, the corresponding FFEA mesh and the 'solid' version of this mesh (Richardson et al, 2014).…”