Enumerating Pathways of Proton Abstraction Based on a Spatial and Electrostatic Analysis of Residues in the Catalytic Site

The pathways of proton abstraction (PA), a key aspect of most catalytic reactions, is often controversial and highly debated. Ultrahigh-resolution diffraction studies, molecular dynamics, quantum mechanics and molecular mechanic simulations are often adopted to gain insights in the PA mechanisms in enzymes. These methods require expertise and effort to setup and can be computationally intensive. We present a push button methodology – Proton abstraction Simulation (PRISM) – to enumerate the possible pathways of PA in a protein with known 3D structure based on the spatial and electrostatic properties of residues in the proximity of a given nucleophilic residue. Proton movements are evaluated in the vicinity of this nucleophilic residue based on distances, potential differences, spatial channels and characteristics of the individual residues (polarity, acidic, basic, etc). Modulating these parameters eliminates their empirical nature and also might reveal pathways that originate from conformational changes. We have validated our method using serine proteases and concurred with the dichotomy in PA in Class A β-lactamases, both of which are hydrolases. The PA mechanism in a transferase has also been corroborated. The source code is made available at www.sanchak.com/prism

Disruption of key NADH-binding pocket residues of the Mycobacterium tuberculosis InhA affects DD-CoA binding ability

Tuberculosis (TB) is a global health problem that affects over 10 million people. There is an urgent need to develop novel antimicrobial therapies to combat TB. To achieve this, a thorough understanding of key validated drug targets is required. The enoyl reductase InhA, responsible for synthesis of essential mycolic acids in the mycobacterial cell wall, is the target for the frontline anti-TB drug isoniazid. To better understand the activity of this protein a series of mutants, targeted to the NADH co-factor binding pocket were created. Residues P193 and W222 comprise a series of hydrophobic residues surrounding the cofactor binding site and mutation of both residues negatively affect InhA function. Construction of an M155A mutant of InhA results in increased affinity for NADH and DD-CoA turnover but with a reduction in Vmax for DD-CoA, impairing overall activity. This suggests that NADH-binding geometry of InhA likely permits long-range interactions between residues in the NADH-binding pocket to facilitate substrate turnover in the DD-CoA binding region of the protein. Understanding the precise details of substrate binding and turnover in InhA and how this may affect protein-protein interactions may facilitate the development of improved inhibitors enabling the development of novel anti-TB drugs

DNA methylation signal has a major role in the response of human breast cancer cells to the microenvironment

International audienceBreast cancer-associated fibroblasts (CAFs) have a crucial role in tumor initiation, metastasis and therapeutic resistance by secreting various growth factors, cytokines, protease and extracellular matrix components. Soluble factors secreted by CAFs are involved in many pathways including inflammation, metabolism, proliferation and epigenetic modulation, suggesting that CAF-dependent reprograming of cancer cells affects a large set of genes. This paracrine signaling has an important role in tumor progression, thus deciphering some of these processes could lead to relevant discoveries with subsequent clinical implications. Here, we investigated the mechanisms underlying the changes in gene expression patterns associated with the cross-talk between breast cancer cells and the stroma. From RNAseq data obtained from breast cancer cell lines grown in presence of CAF-secreted factors, we identified 372 upregulated genes, exhibiting an expression level positively correlated with the stromal content of breast cancer specimens. Furthermore, we observed that gene expression changes were not mediated through significant DNA methylation changes. Nevertheless, CAF-secreted factors but also stromal content of the tumors remarkably activated specific genes characterized by a DNA methylation pattern: hypermethylation at transcription start site and shore regions. Experimental approaches (inhibition of DNA methylation, knockdown of methyl-CpG-binding domain protein 2 and chromatin immunoprecipitation assays) indicated that this set of genes was epigenetically controlled. These data elucidate the importance of epigenetics marks in the cancer cell reprogramming induced by stromal cell and indicated that the interpreters of the DNA methylation signal have a major role in the response of the cancer cells to the microenvironment

Unintended consequences? Water molecules at biological and crystallographic protein–protein interfaces

tThe importance of protein–protein interactions (PPIs) is becoming increasingly appreciated, as theseinteractions lie at the core of virtually every biological process. Small molecule modulators that targetPPIs are under exploration as new therapies. One of the greatest obstacles faced in crystallographicallydetermining the 3D structures of proteins is coaxing the proteins to form “artificial” PPIs that lead touniform crystals suitable for X-ray diffraction. This work compares interactions formed naturally, i.e.,“biological”, with those artificially formed under crystallization conditions or “non-biological”. In partic-ular, a detailed analysis of water molecules at the interfaces of high-resolution (≤2.30˚A) X-ray crystalstructures of protein–protein complexes, where 140 are biological protein–protein complex structuresand 112 include non-biological protein–protein interfaces, was carried out using modeling tools basedon the HINT forcefield. Surprisingly few and relatively subtle differences were observed between thetwo types of interfaces: (i) non-biological interfaces are more polar than biological interfaces, yet there isbetter organized hydrogen bonding at the latter; (ii) biological associations rely more on water-mediatedinteractions with backbone atoms compared to non-biological associations; (iii) aromatic/planar residuesplay a larger role in biological associations with respect to water, and (iv) Lys has a particularly large roleat non-biological interfaces. A support vector machines (SVMs) classifier using descriptors from this studywas devised that was able to correctly classify 84% of the two interface type

The TESS-keck survey. III. A stellar obliquity measurement of TOI-1726 c

We report the measurement of a spectroscopic transit of TOI-1726c, one of two planets transiting a G-type star with V = 6.9 in the Ursa Major Moving Group (∼400 Myr). With a precise age constraint from cluster membership, TOI-1726 provides a great opportunity to test various obliquity excitation scenarios that operate on different timescales. By modeling the Rossiter-McLaughlin (RM) effect, we derived a sky-projected obliquity of -1-+3235∘. This result rules out a polar/retrograde orbit and is consistent with an aligned orbit for planet c. Considering the previously reported, similarly prograde RM measurement of planet b and the transiting nature of both planets, TOI-1726 tentatively conforms to the overall picture that compact multitransiting planetary systems tend to have coplanar, likely aligned orbits. TOI-1726 is also a great atmospheric target for understanding differential atmospheric loss of sub-Neptune planets (planet b 2.2 R☉ and c 2.7 R☉ both likely underwent photoevaporation). The coplanar geometry points to a dynamically cold history of the system that simplifies any future modeling of atmospheric escape

Methylation specific targeting of a chromatin remodeling complex from sponges to humans

DNA cytosine methylation and methyl-cytosine binding domain (MBD) containing proteins are found throughout all vertebrate species studied to date. However, both the presence of DNA methylation and pattern of methylation varies among invertebrate species. Invertebrates generally have only a single MBD protein, MBD2/3, that does not always contain appropriate residues for selectively binding methylated DNA. Therefore, we sought to determine whether sponges, one of the most ancient extant metazoan lineages, possess an MBD2/3 capable of recognizing methylated DNA and recruiting the associated nucleosome remodeling and deacetylase (NuRD) complex. We find that Ephydatia muelleri has genes for each of the NuRD core components including an EmMBD2/3 that selectively binds methylated DNA. NMR analyses reveal a remarkably conserved binding mode, showing almost identical chemical shift changes between binding to methylated and unmethylated CpG dinucleotides. In addition, we find that EmMBD2/3 and EmGATAD2A/B proteins form a coiled-coil interaction known to be critical for the formation of NuRD. Finally, we show that knockdown of EmMBD2/3 expression disrupts normal cellular architecture and development of E. muelleri. These data support a model in which the MBD2/3 methylation-dependent functional role emerged with the earliest multicellular organisms and has been maintained to varying degrees across animal evolution