Exploring protein functions from structural flexibility using CABS-flex modeling.

Understanding protein function often necessitates characterizing the flexibility of protein structures. However, simulating protein flexibility poses significant challenges due to the complex dynamics of protein systems, requiring extensive computational resources and accurate modeling techniques. In response to these challenges, the CABS-flex method has been developed as an efficient modeling tool that combines coarse-grained simulations with all-atom detail. Available both as a web server and a standalone package, CABS-flex is dedicated to a wide range of users. The web server version offers an accessible interface for straightforward tasks, while the standalone command-line program is designed for advanced users, providing additional features, analytical tools, and support for handling large systems. This paper examines the application of CABS-flex across various structure-function studies, facilitating investigations into the interplay among protein structure, dynamics, and function in diverse research fields. We present an overview of the current status of the CABS-flex methodology, highlighting its recent advancements, practical applications, and forthcoming challenges.

Are there double knots in proteins? Prediction and in vitro verification based on TrmD-Tm1570 fusion from C. nitroreducens.

We have been aware of the existence of knotted proteins for over 30 years-but it is hard to predict what is the most complicated knot that can be formed in proteins. Here, we show new and the most complex knotted topologies recorded to date-double trefoil knots (31 #31). We found five domain arrangements (architectures) that result in a doubly knotted structure in almost a thousand proteins. The double knot topology is found in knotted membrane proteins from the CaCA family, that function as ion transporters, in the group of carbonic anhydrases that catalyze the hydration of carbon dioxide, and in the proteins from the SPOUT superfamily that gathers 31 knotted methyltransferases with the active site-forming knot. For each family, we predict the presence of a double knot using AlphaFold and RoseTTaFold structure prediction. In the case of the TrmD-Tm1570 protein, which is a member of SPOUT superfamily, we show that it folds in vitro and is biologically active. Our results show that this protein forms a homodimeric structure and retains the ability to modify tRNA, which is the function of the single-domain TrmD protein. However, how the protein folds and is degraded remains unknown.

Residue conservation elucidates the evolution of r-proteins in ribosomal assembly and function.

Ribosomes are the translational machineries having two unequal subunits, small subunit (SSU) and large subunit (LSU) across all the domains of life. Origin and evolution of ribosome are encoded in its structure, and the core of the ribosome is highly conserved. Here, we have used Shannon entropy to analyze the evolution of ribosomal proteins (r-proteins) across the three domains of life. Moreover, we have analyzed the residue conservation at protein-protein (PP) and protein-RNA (PR) interfaces in SSU and LSU. Furthermore, we have studied the evolution of early, intermediate and late binding r-proteins. We show that the r-proteins of Thermus thermophilus are better conserved during the evolution. Furthermore, we find the late binders are better conserved than the early and the intermediate binders. The residues at the interior of the r-proteins are the most conserved followed by those at the interface and the solvent accessible surface. Additionally, we show that the residues at the PP interfaces are better conserved than those at the PR interfaces. However, between PR and PP interfaces, the multi-interface residues at the former are better conserved than those at the latter ones. Our findings may provide insights into the evolution of r-proteins in ribosomal assembly and function.

Dissecting macromolecular recognition sites in ribosome: implication to its self-assembly.

Interactions between macromolecules play a crucial role in ribosome assembly that follows a highly coordinated process involving RNA folding and binding of ribosomal proteins (r-proteins). Although extensive studies have been carried out to understand macromolecular interactions in ribosomes, most of them are confined to either large or small ribosomal-subunit of few species. A comparative analysis of macromolecular interactions across different domains is still missing. We have analyzed the structural and physicochemical properties of protein-protein (PP), protein-RNA (PR) and RNA-RNA (RR) interfaces in small and large subunits of ribosomes, as well as in between the two subunits. Additionally, we have also developed Random Forest (RF) classifier to catalog the r-proteins. We find significant differences as well as similarities in macromolecular recognition sites between ribosomal assemblies of prokaryotes and eukaryotes. PR interfaces are substantially larger and have more ionic interactions than PP and RR interfaces in both prokaryotes and eukaryotes. PP, PR and RR interfaces in eukaryotes are well packed compared to those in prokaryotes. However, the packing density between the large and the small subunit interfaces in the entire assembly is strikingly low in both prokaryotes and eukaryotes, indicating the periodic association and dissociation of the two subunits during the translation. The structural and physicochemical properties of PR interfaces are used to predict the r-proteins in the assembly pathway into early, intermediate and late binders using RF classifier with an accuracy of 80%. The results provide new insights into the classification of r-proteins in the assembly pathway.

Dissecting protein-protein interactions in proteasome assembly: Implication to its self-assembly.

The 26S proteasome is a multi-catalytic ATP-dependent protease complex that recognizes and cleaves damaged or misfolded proteins to maintain cellular homeostasis. The 26S subunit consists of 20S core and 19S regulatory particles. 20S core particle consists of a stack of heptameric alpha and beta subunits. To elucidate the structure-function relationship, we have dissected protein-protein interfaces of 20S core particle and analyzed structural and physiochemical properties of intra-alpha, intra-beta, inter-beta, and alpha-beta interfaces. Furthermore, we have studied the evolutionary conservation of 20S core particle. We find the size of intra-alpha interfaces is significantly larger and is more hydrophobic compared with other interfaces. Inter-beta interfaces are well packed, more polar, and have higher salt-bridge density than other interfaces. In proteasome assembly, residues in beta subunits are better conserved than alpha subunits, while multi-interface residues are the most conserved. Among all the residues at the interfaces of both alpha and beta subunits, Gly is highly conserved. The largest size of intra-alpha interfaces complies with the hypothesis that large interfaces form first during the 20S assembly. The tight packing of inter-beta interfaces makes the core particle impenetrable from outer wall of the cylinder. Comparing the three domains, eukaryotes have large and well-packed interfaces followed by archaea and bacteria. Our findings provide a structural basis of assembly of 20S core particle in all the three domains of life.

Molecular architecture of protein-RNA recognition sites.

The molecular architecture of protein-RNA interfaces are analyzed using a non-redundant dataset of 152 protein-RNA complexes. We find that an average protein-RNA interface is smaller than an average protein-DNA interface but larger than an average protein-protein interface. Among the different classes of protein-RNA complexes, interfaces with tRNA are the largest, while the interfaces with the single-stranded RNA are the smallest. Significantly, RNA contributes more to the interface area than its partner protein. Moreover, unlike protein-protein interfaces where the side chain contributes less to the interface area compared to the main chain, the main chain and side chain contributions flipped in protein-RNA interfaces. We find that the protein surface in contact with the RNA in protein-RNA complexes is better packed than that in contact with the DNA in protein-DNA complexes, but loosely packed than that in contact with the protein in protein-protein complexes. Shape complementarity and electrostatic potential are the two major factors that determine the specificity of the protein-RNA interaction. We find that the H-bond density at the protein-RNA interfaces is similar with that of protein-DNA interfaces but higher than the protein-protein interfaces. Unlike protein-DNA interfaces where the deoxyribose has little role in intermolecular H-bonds, due to the presence of an oxygen atom at the 2' position, the ribose in RNA plays significant role in protein-RNA H-bonds. We find that besides H-bonds, salt bridges and stacking interactions also play significant role in stabilizing protein-nucleic acids interfaces; however, their contribution at the protein-protein interfaces is insignificant.