Discovery of a carbonic anhydrase-Rubisco supercomplex within the alpha-carboxysome

Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO2 fixation, Rubisco and carbonic anhydrase, and are the centerpiece of the bacterial CO2 concentrating mechanism (CCM). In the CCM, actively accumulated cytosolic bicarbonate diffuses into the carboxysome and is converted to CO2 by carbonic anhydrase, producing a high CO2 concentration near Rubisco and ensuring efficient carboxylation. Self-assembly of the α-carboxysome is orchestrated by the intrinsically disordered scaffolding protein, CsoS2, which interacts with both Rubisco and carboxysomal shell proteins, but it is unknown how CsoSCA, the carbonic anhydrase, is incorporated into the α-carboxysome. Here, we present the structural basis of carbonic anhydrase encapsulation into α-carboxysomes from Halothiobacillus neapolitanus. We find that CsoSCA interacts directly with Rubisco via an intrinsically disordered N-terminal domain. A 1.98 Å single-particle cryo-electron microscopy structure of Rubisco in complex with this peptide reveals that CsoSCA binding is predominantly mediated by a network of hydrogen bonds. CsoSCAs binding site overlaps with that of CsoS2 but the two proteins utilize substantially different motifs and modes of binding, revealing a plasticity of the Rubisco binding site. Our results advance the understanding of biogenesis of carboxysomes and highlights the importance of Rubisco, not only as an enzyme, but also as a hub protein central for assembling supercomplexes.

Molecular Dynamics Simulations of resistin and RELMβ proteins: Insight into structural dynamics

Diabetes mellitus and high levels of resistin are risk factors for COVID-19, suggest- ing a shared mechanism for their contribution to the increased severity of COVID-19. Resistin belongs to the family of resistin-like molecules (RELMs) whose implications for inflammatory and metabolic dysfunctions warrant its study in order to shed light on the etiology of these concerning pathologies. In this work, our objective is to char- acterize the structural dynamics of the reported crystallized resistin-like molecules. We performed molecular dynamics simulations of all-atom solvated protein at physiological and high temperatures for the three mouse structures reported so far. We found that in all the structures studied, there is a loss of helicity as a first step of protein denat- uration. There is a high stability of the globular β-sheet domain in resistin protein structures that is not conserved for RELMβ. At high temperature, we found a partial interconversion of α-helices into β-sheets in all proteins, indicating that this propensity is not only found during aggregation but also heating. We had been able to identify a largely persistent hydrogen-bond network shared by all the proteins in the interchain globular domain at room temperature. This network of hydrogen bonds is conserved considerably at high temperature in resistin structures, but not in RELMβ. These findings may guide future studies to increase our understanding of the different and shared mechanisms of action of RELMs.

The Mixed Hydrogen Bonds Network in a Liquid System Ethylene Glycol-Monoethanolamine

The mixed network of hydrogen bonds in the ethylene glycol (EG) - monoethanolamine (MEA) system is described by molecular dynamics (MD) methods, graph theory, and Delaunay simplexes at 300 K in the entire concentration range. It is shown that at low MEA concentrations, all molecules in the system are linked into a spatial network of H-bonds; at high MEA concentrations, this number is 96%. Detailed characteristics of the networks are given. The resulting picture is expanded by studying the system using the Delaunay simplex method. The calculations are compared for different charges on the atoms of the MEA molecule.

IR-Spectroscopy of Aqueous Solutions of Monoethanolamine

The monoethanolamine (MEA)-water system has been studied by IR spectroscopy and quantum-chemical calculations (DFT B3LYP). It was found that spatial networks both of water and MEA are continuously rearranging depending on the content of the system components. Water molecules are embedded into the net of MEA, and molecules of MEA into the water net, thereby forming a mixed network of hydrogen bonds.

Correlation of Translational and Rotational Motion of Water Molecules in Molecular Dynamic Models

The motion of a rigid molecule in a computer model can be described as the sum of the displacement of the center of mass of the molecule and the rotation of the molecule around an axis passing through this center. The interaction of a molecule with its environment leads to a correlation between these movements. Earlier in [1], we studied the distributions of angles between the directions of the displacement vectors and the axes of rotation of water molecules, as well as between them and the internal vectors of these molecules. This paper describes the correlation of the magnitudes of these displacements, that is, the lengths of the displacement vectors and angles of accompanying rotations. The correlation coefficients of these characteristics are calculated for intervals of different durations at different temperatures and pressures, and the character times of preservation of these correlations are determined. We believe that the times found by us represent the lifetimes of the local environments of molecules. For water molecules, a change in the local environment is accompanied by a change in the structure of the nearest section of the network of hydrogen bonds, and therefore the lifetimes of local environments are close to the lifetimes of these bonds.

Thermophilic Enzymes

Substantial improvements in the industrial production of goods led to a widespread feeling of unlimited access to food, commodities, and energy. As greener alternatives for industrial processes are in demand, scientists have turned to enzymes, looking for apt biocatalysts. Focusing on extremophiles, this mini review draws a comparison between thermophiles and their mesophilic counterparts, exploring what features are instrumental to their thermostability. A higher number of ion-pairs, hydrophobicity of buried side chains, compact tertiary structure cores, and a complex network of hydrogen bonds are the four main characteristics responsible for the robustness of thermophilic enzymes.

Molecular Dynamics Studies of Therapeutic Liquid Mixtures and Their Binding to Mycobacteria

Tuberculosis is an highly contagious disease still considered by the WHO as one of most infectious diseases worldwide. The therapeutic approach, used to prevent and treat tuberculosis targets the Mycobacterium tuberculosis complex, comprises a combination of drugs administrated for long periods of time, which, in many cases, could cause several adverse effects and, consequently, low compliance of the patient to the treatment and drug-resistance. Therefore, therapeutic liquid mixtures formulated with anti-tuberculosis drugs and/or adjuvants in tuberculosis therapy are an interesting approach to prevent toxic effects and resistance to anti-tuberculosis drugs. The herein formulated therapeutic liquid mixtures, including ethambutol, arginine, citric acid and water under different molar ratios, were studied through a molecular dynamics approach to understand how ethambutol and arginine could be stabilized by the presence of citric acid and/or water in the mixture. To gain insights on how the uptake of these mixtures into the mycobacteria cell may occur and how a mycobacterial ABC transporter could contribute to this transport, multiple simultaneous ligand docking was performed. Interactions between citric acid and ethambutol involving the carboxyl and hydroxyl groups of citric acid with the amines of ethambutol were identified as the most critical ones. Water molecules present in the mixture provides the necessary network of hydrogen bonds that stabilize the mixture. Molecular docking additionally provided an interesting hypothesis on how the different mixture components may favor binding of ethambutol to an ABC importer. The data presented in this work helps to better understand these mixtures as well as to provide cues on the mechanisms that allow them to cross the mycobacterial cell membrane.

Allosteric communication in DNA polymerase clamp loaders relies on a critical hydrogen-bonded junction

Clamp loaders are AAA+ ATPases that load sliding clamps onto DNA. We mapped the mutational sensitivity of the T4 bacteriophage sliding clamp and clamp loader by deep mutagenesis, and found that residues not involved in catalysis or binding display remarkable tolerance to mutation. An exception is a glutamine residue in the AAA+ module (Gln 118) that is not located at a catalytic or interfacial site. Gln 118 forms a hydrogen-bonded junction in a helical unit that we term the central coupler, because it connects the catalytic centers to DNA and the sliding clamp. A suppressor mutation indicates that hydrogen bonding in the junction is important, and molecular dynamics simulations reveal that it maintains rigidity in the central coupler. The glutamine-mediated junction is preserved in diverse AAA+ ATPases, suggesting that a connected network of hydrogen bonds that links ATP molecules is an essential aspect of allosteric communication in these proteins.