SARS-CoV-2 encodes four structural proteins incorporated into virions, spike (S), envelope (E), nucleocapsid (N), and membrane (M). M plays an essential role in viral assembly by organizing other ...structural proteins through physical interactions and directing them to sites of viral budding. As the most abundant protein in the viral envelope and a target of patient antibodies, M is a compelling target for vaccines and therapeutics. Still, the structure of M and molecular basis for its role in virion formation are unknown. Here, we present the cryo-EM structure of SARS-CoV-2 M in lipid nanodiscs to 3.5 Å resolution. M forms a 50 kDa homodimer that is structurally related to the SARS-CoV-2 ORF3a viroporin, suggesting a shared ancestral origin. Structural comparisons reveal how intersubunit gaps create a small, enclosed pocket in M and large open cavity in ORF3a, consistent with a structural role and ion channel activity, respectively. M displays a strikingly electropositive cytosolic surface that may be important for interactions with N, S, and viral RNA. Molecular dynamics simulations show a high degree of structural rigidity in a simple lipid bilayer and support a role for M homodimers in scaffolding viral assembly. Together, these results provide insight into roles for M in coronavirus assembly and structure.
A wide range of mutations in the kinesin motor Kif5A have been linked to a neuronal disorder called hereditary spastic paraplegia (HSP). The position of these mutations can vary, and a range of ...different motile behaviors have been observed, indicating that the HSP mutants can alter distinct aspects of kinesin mechanochemistry. While focusing on four key HSP-associated mutants, this study examined the structural and dynamic perturbations that arise from these mutations using a series of different computational methods, ranging from bioinformatics analyses to all-atom simulations, that account for solvent effects explicitly. We show that two catalytic domain mutations (R280S and K253N) reduce the microtubule (MT) binding affinity of the kinesin head domains appreciably, while N256S has a much smaller impact. Bioinformatics analysis suggests that the stalk mutation A361V perturbs motor dimerization. Subsequent integration of these effects into a coarse-grained structure-based model of dimeric kinesin revealed that the order–disorder transition of the neck linker is substantially affected, indicating a hampered directionality and processivity of kinesin. The present analyses therefore suggest that, in addition to kinesin-MT binding and coiled-coil dimerization, HSP mutations affecting motor stepping transitions and processivity can lead to disease.
Cytoplasmic dynein, an AAA+ motif containing motor, generates force and movement along the microtubule to execute important biological functions including intracellular material transport and cell ...division by hydrolyzing ATP. Among the six AAA+ domains, AAA1 is the primary ATPase site where a single ATP hydrolysis generates a single step. Nucleotide states in AAA3 gate dynein’s activity, suggesting that AAA3 acts as a regulatory switch. However, the comprehensive structural perspective of AAA3 in dynein’s mechanochemical cycle remains unclear. Here, we explored the allosteric transition path of dynein involving AAA3 using a coarse-grained structure-based model. ATP binding to the AAA1 domain creates a cascade of conformational changes through the other domains of the ring, which leads to the pre-power stroke formation. The linker domain, which is the mechanical element of dynein, shifts from a straight to a bent conformation during this process. In our present study, we observe that AAA3 gates the allosteric communication from AAA1 to the microtubule binding domain (MTBD) through AAA4 and AAA5. The MTBD is linked to the AAA+ ring via a coiled-coil stalk and a buttress domain, which are extended from AAA4 and AAA5, respectively. Further analysis also uncovers the role of AAA3 in the linker movement. The free energy calculation shows that the linker prefers the straight conformation when AAA3 remains in the ATP-bound condition. As AAA3 restricts the motion of AAA4 and AAA5, the linker/AAA5 interactions get stabilized, and the linker cannot move to the pre-power stroke state that halts the complete structural transition required for the mechanochemical cycle. Therefore, we suggest that AAA3 governs dynein’s mechanochemical cycle and motility by controlling the AAA4 and AAA5 domains that further regulate the linker movement and the power stroke formation.
Dyneins, a class of motor proteins consisting of six AAA+ modules (AAA1-AAA6), convert chemical energy derived from the hydrolysis of ATP into mechanical energy to walk along the microtubule track ...towards its minus end while accomplishing various cellular tasks including the transportation of various intracellular cargos. In a full mechanochemical cycle, dynein goes through ATP binding induced open to closed state transition of AAA1, hydrolysis of that ATP and closed to open state transition induced by the release of hydrolysed products along with linker remodelling in different nucleotide states. Here we built structure based models (SBMs) to explore the sequence of events of this mechanochemical cycle from structural aspects. Free energy and kinetic simulation approaches on a multi-basin SBM of dynein reveal the following pathways: (1) in the closing pathway, the AAA1 domain first converts to a closed state followed by the movement of the linker and (2) in the opening transition, initially the AAA1 domain partially opens up and then the complete linker movement takes place followed by the complete opening of the AAA1 domain. In the opening transition, we have observed two intermediate states from our simulations where the AAA1 domain is partially opened. However, in one state the linker is at a closed position and in the other the linker is at an open position. The existence of such intermediates (Pi released, ADP bound state) of dynein has been suggested by numerous experimental studies earlier. Finally, we discuss the biological relevance of this sequence of events in terms of processivity and efficiency of the cycle. The current study also shows how the basic principle of protein folding can be extended to understand complex phenomena like the stepping mechanism of motor proteins.
Dynein, a large and complex motor protein, harnesses energy from adenosine triphosphate (ATP) hydrolysis to regulate essential cellular activities. The ATP hydrolysis mechanism for the dynein motor ...is still shrouded in mystery. Herein, molecular dynamics simulations of a dynein motor disclosed that two water molecules are present close to the γ-phosphate of ATP and Glu1742 at the AAA1 site of dynein. We have proposed three possible mechanisms for the ATP hydrolysis. We divulge by using a quantum mechanics/molecular mechanics (QM/MM) study that two water molecules and Glu1742 are crucial for facilitating the ATP hydrolysis reaction in dynein. Moreover, the ATP hydrolysis step is initiated by the activation of lytic water (W1) by Glu1742 through relay proton transfers with the help of auxiliary water (W2) yielding HPO
and ADP, as a product. In the next step, a proton is shifted back from Glu1742 to generate inorganic phosphate (H
PO
) via another relay proton transfer event. The overall activation barrier for the Glu1742 assisted ATP hydrolysis is found to be the most favourable pathway compared to other plausible pathways. We also unearthed that ATP hydrolysis in dynein follows a so-called associative-like pathway in its rate-limiting step. Our study ascertained the important indirect roles of the two amino acids (such as Arg2109, Asn1792) and Mg
ion in the ATP hydrolysis of dynein. Additionally, multiple sequence alignment of the different organisms of dynein motors has conveyed the evolutionary importance of the Glu1742, Asn1742, and Arg2109 residues, respectively. As similar mechanisms are also prevalent in other motors, and GTPase and ATPase enzymes, the present finding spells out the definitive requirement of a proton relay process through an extended water-chain as one of the key components in an enzymatic ATP hydrolysis reaction.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a lipid-enveloped virus that acquires its lipid bilayer from the host cell it infects. SARS-CoV-2 can spread from cell to cell or from ...patient to patient by undergoing assembly and budding to form new virions. The assembly and budding of SARS-CoV-2 is mediated by several structural proteins known as envelope (E), membrane (M), nucleoprotein (N), and spike (S), which can form virus-like particles (VLPs) when co-expressed in mammalian cells. Assembly and budding of SARS-CoV-2 from the host ER-Golgi intermediate compartment is a critical step in the virus acquiring its lipid bilayer. To date, little information is available on how SARS-CoV-2 assembles and forms new viral particles from host membranes. In this study, we used several lipid binding assays and found the N protein can strongly associate with anionic lipids including phosphoinositides and phosphatidylserine. Moreover, we show lipid binding occurs in the N protein C-terminal domain, which is supported by extensive in silico analysis. We demonstrate anionic lipid binding occurs for both the free and the N oligomeric forms, suggesting N can associate with membranes in the nucleocapsid form. Based on these results, we present a lipid-dependent model based on in vitro, cellular, and in silico data for the recruitment of N to assembly sites in the lifecycle of SARS-CoV-2.
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