Conformational flexibility of the oncogenic protein LMO2 primes the formation of the multi-protein transcription complex.

LMO2 was discovered via chromosomal translocations in T-cell leukaemia and shown normally to be essential for haematopoiesis. LMO2 is made up of two LIM only domains (thus it is a LIM-only protein) and forms a bridge in a multi-protein complex. We have studied the mechanism of formation of this complex using a single domain antibody fragment that inhibits LMO2 by sequestering it in a non-functional form. The crystal structure of LMO2 with this antibody fragment has been solved revealing a conformational difference in the positioning and angle between the two LIM domains compared with its normal binding. This contortion occurs by bending at a central helical region of LMO2. This is a unique mechanism for inhibiting an intracellular protein function and the structural contusion implies a model in which newly synthesized, intrinsically disordered LMO2 binds to a partner protein nucleating further interactions and suggests approaches for therapeutic targeting of LMO2.

Hexameric molecular motors: P4 packaging ATPase unravels the mechanism.

Genome packaging into an empty capsid is an essential step in the assembly of many complex viruses. In double-stranded RNA (dsRNA) bacteriophages of the Cystoviridae family this step is performed by a hexameric helicase P4 which is one of the simplest packaging motors found in nature. Biochemical and structural studies of P4 proteins have led to a surprising finding that these proteins bear mechanistic and structural similarities to a variety of the pervasive RecA/F1-ATPase-like motors that are involved in diverse biological functions. This review describes the role of P4 proteins in assembly, transcription and replication of dsRNA bacteriophages as it has emerged over the past decade while focusing on the most recent structural studies. The P4 mechanism is compared with the models proposed for the related hexameric motors.

Supplanting crystallography or supplementing microscopy? A combined approach to the study of an enveloped virus.

The recent advances in the resolution obtained by single-particle reconstructions from cryo-electron microscopy (cryo-EM) have led to an increase in studies that combine X-ray crystallographic results with those of electron microscopy (EM). Here, such a combination is described in the determination of the structure of an enveloped animal virus, Semliki Forest virus, at 9 A resolution. The issues of model bias in determination of the structure, the definition of resolution in a single-particle reconstruction, the effect of the correction of the contrast-transfer function on the structure determined and the use of a high-resolution structure of a subunit in the interpretation of the structure of the complex are addressed.

Cryo-electron microscopy reveals the functional organization of an enveloped virus, Semliki Forest virus.

Semliki Forest virus serves as a paradigm for membrane fusion and assembly. Our icosahedral reconstruction combined 5276 particle images from 48 cryo-electron micrographs and determined the virion structure to 9 A resolution. The improved resolution of this map reveals an N-terminal arm linking capsid subunits and defines the spike-capsid interaction sites. It illustrates the paired helical nature of the transmembrane segments and the elongated structures connecting them to the spike projecting domains. A 10 A diameter density in the fusion protein lines the cavity at the center of the spike. These clearly visible features combine with the variation in order between the layers to provide a framework for understanding the structural changes during the life cycle of an enveloped virus.

The first step: activation of the Semliki Forest virus spike protein precursor causes a localized conformational change in the trimeric spike.

The structure of the particle formed by the SFVmSQL mutant of Semliki Forest virus (SFV) has been defined by cryo-electron microscopy and image reconstruction to a resolution of 21 A. The SQL mutation blocks the cleavage of p62, the precursor of the spike proteins E2 and E3, which normally occurs in the trans-Golgi. The uncleaved spike protein is insensitive to the low pH treatment that triggers membrane fusion during entry of the wild-type virus. The conformation of the spike in the SFVmSQL particle should correspond to that of the inactive precursor found in the early stages of the secretory pathway. Comparison of this "precursor" structure with that of the mature, wild-type, virus allows visualization of the changes that lead to activation, the first step in the pathway toward fusion. We find that the conformational change in the spike is dramatic but localized. The projecting domains of the spikes are completely separated in the precursor and close to generate a cavity in the mature spike. E1, the fusion peptide-bearing protein, interacts only with the p62 in its own third of the trimer before cleavage and then collapses to form a trimer of heterotrimers (E1E2E3)3 surrounding the cavity, poised for the pH-induced conformational change that leads to fusion. The capsid, transmembrane regions and the spike skirts (thin layers of protein that link spikes above the membrane) remain unchanged by cleavage. Similarly, the interactions of the spikes with the nucleocapsid through the transmembrane domains remain constant. Hence, the interactions that lead to virus assembly are unaffected by the SFVmSQL mutation.

High-resolution icosahedral reconstruction: fulfilling the promise of cryo-electron microscopy.

Two recent papers have defined the secondary structure of the hepatitis virus capsid using a combination of cryo-electron microscopy and icosahedral image reconstruction. These two papers do more than reveal a new fold for a virus protein; they herald a new era in which image reconstruction of single particles will provide reliable high-resolution structural information. In revealing the promise of these techniques to the structural biology community, their two papers should play a seminal role for single particle work, similar to that of the work of Unwin and Henderson on bacteriorhodopsin in revealing the potential of electron microscopy of membrane protein crystals. Indeed, the success of these single particle methods owes much to the development of high-resolution techniques for two-dimensional crystals. This review will summarize some of the history of icosahedral reconstruction from cryo-electron micrographs, compare the two different approaches used to obtain the recent results and outline some of the challenges and promises for the future.