Hydrogen-bonding networks and RNA bases revealed by cryo electron microscopy suggest a triggering mechanism for calcium switches

Abstract

Helical assemblies such as filamentous viruses, flagella, and F-actin represent an important category of structures in biology. As the first discovered virus, tobacco mosaic virus (TMV) was at the center of virus research. Previously, the structure of TMV was solved at atomic detail by X-ray fiber diffraction but only for its dormant or high-calcium-concentration state, not its low-calcium-concentration state, which is relevant to viral assembly and disassembly inside host cells. Here we report a helical reconstruction of TMV in its calcium-free, metastable assembling state at 3.3 Å resolution by cryo electron microscopy, revealing both protein side chains and RNA bases. An atomic model was built de novo showing marked differences from the high-calcium, dormant-state structure. Although it could be argued that there might be inaccuracies in the latter structure derived from X-ray fiber diffraction, these differences can be interpreted as conformational changes effected by calcium-driven switches, a common regulatory mechanism in plant viruses. Our comparisons of the structures of the low-and highcalcium states indicate that hydrogen bonds formed by Asp116 and Arg92 in the place of the calcium ion of the dormant (highcalcium) state might trigger allosteric changes in the RNA basebinding pockets of the coat protein. In turn, the coat protein-RNA interactions in our structure favor an adenine-X-guanine (A*G) motif over the G*A motif of the dormant state, thus offering an explanation underlying viral assembly initiation by an AAG motif.H elical assemblies represent a very important class of biological structures, performing various tasks for the proper functions of life or executing malicious functions in disease. For example, protein helical assemblies such as cytoskeletal networks and muscle fibers are essential components in our bodies (1, 2). Other helical assemblies include bacterial flagella and secretion systems. Helical viruses, such as tobacco mosaic virus (TMV), account for a major fraction of the virus kingdom. Such helical structures, bearing a special kind of 2D periodicity and potential flexibility, are difficult for structural determination to atomic resolution by conventional methods including X-ray crystallography and NMR. Thus, the molecular mechanisms of action in many of these systems are not understood to atomic detail. Even for TMV, the first virus ever isolated and extensively studied (3) by both X-ray diffraction (e.g., 4-8) and cryo electron microscopy (cryoEM) (9, 10), we still do not know its structure at atomic detail in a state that is relevant to its assembly and disassembly processes, and the underlying mechanisms of these processes remain unknown.The viral RNA of TMV is infectious by itself, but RNA on its own is very unstable. The addition of a 17-kDa coat protein (CP) around the RNA protects the RNA from degrading and makes TMV very stable. The assembly of a rod-shaped virion begins at a two-turn helical CP complex, called the 20S aggregate (11), which recognizes the initiation moti...

Crystal Structure of the Coat Protein of the Flexible Filamentous Papaya Mosaic Virus

“…8 Most recently, several groups have determined the structures of TMV by cryo-electron microscopy (EM) at 4.7 Å resolution, 9 4.6 Å resolution, 10 and 3.3 Å resolution. 11 The tobamoviruses are homologous in sequence identity (40-50%) and share the same four-α-helixbundle fold.…”

Nanoscale device architectures derived from biological assemblies: The case of tobacco mosaic virus and (apo)ferritin

“…2(a)-2(c)]. Its structure, determined by X-ray diffraction 62,63) and later confirmed by cryo-TEM at sub-nm resolution, 64,65) comprises 2130 identical CPs, each consisting of 158 amino acids with acetylated N-termini, when the virions are produced in plants. The CPs in TMV self-organize to form a right-handed helix with 49 subunits in three turns and a pitch of 2.29 nm, 66) corresponding to the distance between two adjacent proteins along the main tube axis [ Fig.…”

Twisted X-Rays: Incoming Waveforms Yielding Discrete Diffraction Patterns for Helical Structures

“…We consider two examples, a (6,5)-Carbon nanotube and TMV virus. The parameters are as in Example 1 respectively 2 of Section 10, with the TMV atomic positions taken from the Protein Data Bank, PDB ID 3J06 [GZ11]. We use the electromagnetic model (98), (99), (24) to calculate the signal from structures of finite length:…”