The structure of a plant-specific partitivirus capsid reveals a unique coat protein domain architecture with an intrinsically disordered protrusion

Persistent plant viruses may be the most common viruses in wild plants. A growing body of evidence for mutualism between such viruses and their hosts, suggests that they play an important role in ecology and agriculture. Here we present the capsid structure of a plant-specific partitivirus, Pepper cryptic virus 1, at 2.9 Å resolution by Cryo-EM. Structural features, including the T = 1 arrangement of 60 coat protein dimers, are shared with fungal partitiviruses and the picobirnavirus lineage of dsRNA viruses. However, the topology of the capsid is markedly different with protrusions emanating from, and partly comprising, the binding interface of coat protein dimers. We show that a disordered region at the apex of the protrusion is not required for capsid assembly and represents a hypervariable site unique to, and characteristic of, the plant-specific partitiviruses. These results suggest a structural basis for the acquisition of additional functions by partitivirus coat proteins that enables mutualistic relationships with diverse plant hosts.

Understanding SARS-CoV-2 budding through molecular dynamics simulations of M and E protein complexes

SARS-CoV-2 and other coronaviruses pose a major threat to global health, yet treatment efforts have largely ignored the process of envelope assembly, a key part of the coronaviral life cycle. When expressed together, the M and E proteins are sufficient to facilitate coronavirus envelope assembly. Envelope assembly leads to budding of coronavirus particles into the ER-Golgi intermediate compartment (ERGIC) and subsequent maturation of the virus, yet the mechanisms behind the budding process remain poorly understood. Better understanding of budding may enable new types of antiviral therapies. To this end, we ran atomistic molecular dynamics (MD) simulations of SARS-CoV-2 envelope assembly using the Feig laboratory's refined structural models of the M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of virtual ERGIC membrane. By examining how these proteins induce membrane curvature in silico, we have obtained insights around how the budding process may occur. In our simulations, M protein dimers acted cooperatively to induce membrane curvature. By contrast, E protein pentamers kept the membrane planar. These results could help guide the development of novel antiviral therapeutics which inhibit coronavirus budding.

Distance-based Reconstruction of Protein Quaternary Structures from Inter-Chain Contacts

Predicting the quaternary structure of protein complex is an important problem. Inter-chain residue-residue contact prediction can provide useful information to guide the ab initio reconstruction of quaternary structures. However, few methods have been developed to build quaternary structures from predicted inter-chain contacts. Here, we introduce a gradient descent optimization algorithm (GD) to build quaternary structures of protein dimers utilizing inter-chain contacts as distance restraints. We evaluate GD on several datasets of homodimers and heterodimers using true or predicted contacts. GD consistently performs better than a simulated annealing method and a Markov Chain Monte Carlo simulation method. Using true inter-chain contacts as input, GD can reconstruct high-quality structural models for homodimers and heterodimers with average TM-score ranging from 0.92 to 0.99 and average interface root mean square distance (I-RMSD) from 0.72 Å to 1.64 Å. On a dataset of 115 homodimers, using predicted inter-chain contacts as input, the average TM-score of the structural models built by GD is 0.76. For 46% of the homodimers, high-quality structural models with TM-score >= 0.9 are reconstructed from predicted contacts. There is a strong correlation between the quality of the reconstructed models and the precision and recall of predicted contacts. If the precision or recall of predicted contacts is >20%, GD can reconstruct good models for most homodimers, indicating only a moderate precision or recall of inter-chain contact prediction is needed to build good structural models for most homodimers. Moreover, the accuracy of reconstructed models positively correlates with the contact density in dimers.

Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells

Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.

Distance-based Reconstruction of Protein Quaternary Structures from Inter-Chain Contacts

Predicting the quaternary structure of a protein complex is an important and challenging problem. Inter-chain residue-residue contact prediction can provide useful information to guide the ab initio reconstruction of quaternary structures of protein complexes. However, few methods have been developed to build quaternary structures from predicted inter-chain contacts. Here, we introduce a new gradient descent optimization algorithm (GD) to build quaternary structures of protein dimers utilizing inter-chain contacts as distance restraints. We evaluate GD on several datasets of homodimers and heterodimers using true or predicted contacts. GD consistently performs better than a simulated annealing method and a Markov Chain Monte Carlo simulation method. Using true inter-chain contacts as input, GD can reconstruct high-quality structural models for homodimers and heterodimers with average TM-score ranging from 0.92 to 0.99 and average interface root mean square distance (I-RMSD) from 0.72 Å to 1.64 Å. On a dataset of 115 homodimers, using predicted inter-chain contacts as input, the average TM-score of the structural models built by GD is 0.76. For 46% of the homodimers, high-quality structural models with TM-score >= 0.9 are reconstructed from predicted contacts. There is a strong correlation between the quality of the reconstructed models and the precision and recall of predicted contacts. If the precision or recall of predicted contacts is >20%, GD can reconstruct good models for most homodimers, indicating only a moderate precision or recall of inter-chain contact prediction is needed to build good structural models for most homodimers. Moreover, the accuracy of reconstructed models positively correlates with the contact density in dimers and depends on the initial model and the probability threshold of selecting predicted contacts for the distance-based structure optimization.

Structural and Functional Peculiarities of Cytoplasmic Tropomyosin Isoforms, the Products of TPM1 and TPM4 Genes

Tropomyosin (Tpm) is one of the major protein partners of actin. Tpm molecules are α-helical coiled-coil protein dimers forming a continuous head-to-tail polymer along the actin filament. Human cells produce a large number of Tpm isoforms that are thought to play a significant role in determining actin cytoskeletal functions. Even though the role of these Tpm isoforms in different non-muscle cells is more or less studied in many laboratories, little is known about their structural and functional properties. In the present work, we have applied various methods to investigate the properties of five cytoplasmic Tpm isoforms (Tpm1.5, Tpm 1.6, Tpm1.7, Tpm1.12, and Tpm 4.2), which are the products of two different genes, TPM1 and TPM4, and also significantly differ by alternatively spliced exons: N-terminal exons 1a2b or 1b, internal exons 6a or 6b, and C-terminal exons 9a, 9c or 9d. Our results demonstrate that structural and functional properties of these Tpm isoforms are quite different depending on sequence variations in alternatively spliced regions of their molecules. The revealed differences can be important in further studies to explain why various Tpm isoforms interact uniquely with actin filaments, thus playing an important role in the organization and dynamics of the cytoskeleton.