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The B7 family represents one of the best-studied subgroups within the Ig superfamily, yet new interactions continue to be discovered. However, this binding promiscuity represents a major challenge for defining the biological contribution of each specific interaction. We developed a strategy for addressing these challenges by combining cell microarray and high-throughput FACS methods to screen for promiscuous binding events, map binding interfaces, and generate functionally selective reagents. Applying this approach to the interactions of mPD-L1 with its receptor mPD-1 and its ligand mB7-1, we identified the binding interface of mB7-1 on mPD-L1 and as a result generated mPD-L1 mutants with binding selectivity for mB7-1 or mPD-1. Next, using a panel of mB7-1 mutants, we mapped the binding sites of mCTLA-4, mCD28 and mPD-L1. Surprisingly, the mPD-L1 binding site mapped to the dimer interface surface of mB7-1, placing it distal from the CTLA-4/CD28 recognition surface. Using two independent approaches, we demonstrated that mPD-L1 and mB7-1 bind in cis, consistent with recent reports from Chaudhri A et al. and Sugiura D et al. We further provide evidence that while CTLA-4 and CD28 do not directly compete with PD-L1 for binding to B7-1, they can disrupt the cis PD-L1:B7-1 complex by reorganizing B7-1 on the cell surface. These observations offer new functional insights into the regulatory mechanisms associated with this group of B7 family proteins and provide new tools to elucidate their function in vitro and in vivo.

The rapid advance in genome sequencing presents substantial challenges for protein functional assignment, with half or more of new protein sequences inferred from these genomes having uncertain assignments. The assignment of enzyme function in functionally diverse superfamilies represents a particular challenge, which we address through a combination of computational predictions, enzymology, and structural biology. Here we describe the results of a focused investigation of a group of enzymes in the enolase superfamily that are involved in epimerizing dipeptides. The first members of this group to be functionally characterized were Ala-Glu epimerases in Eschericiha coli and Bacillus subtilis, based on the operon context and enzymological studies; these enzymes are presumed to be involved in peptidoglycan recycling. We have subsequently studied more than 65 related enzymes by computational methods, including homology modeling and metabolite docking, which suggested that many would have divergent specificities;, i.e., they are likely to have different (unknown) biological roles. In addition to the Ala-Phe epimerase specificity reported previously, we describe the prediction and experimental verification of: (i) a new group of presumed Ala-Glu epimerases; (ii) several enzymes with specificity for hydrophobic dipeptides, including one from Cytophaga hutchinsonii that epimerizes D-Ala-D-Ala; and (iii) a small group of enzymes that epimerize cationic dipeptides. Crystal structures for certain of these enzymes further elucidate the structural basis of the specificities. The results highlight the potential of computational methods to guide experimental characterization of enzymes in an automated, large-scale fashion.

The rate of protein evolution is determined by a combination of selective pressure on protein function and biophysical constraints on protein folding and structure. Determining the relative contributions of these properties is an unsolved problem in molecular evolution with broad implications for protein engineering and function prediction. As a case study, we examined the structural divergence of the rapidly evolving o -succinylbenzoate synthase (OSBS) family, which catalyzes a step in menaquinone synthesis in diverse microorganisms and plants. On average, the OSBS family is much more divergent than other protein families from the same set of species, with the most divergent family members sharing <15% sequence identity. Comparing 11 representative structures revealed that loss of quaternary structure and large deletions or insertions are associated with the family’s rapid evolution. Neither of these properties has been investigated in previous studies to identify factors that affect the rate of protein evolution. Intriguingly, one subfamily retained a multimeric quaternary structure and has small insertions and deletions compared with related enzymes that catalyze diverse reactions. Many proteins in this subfamily catalyze both OSBS and N -succinylamino acid racemization (NSAR). Retention of ancestral structural characteristics in the NSAR/OSBS subfamily suggests that the rate of protein evolution is not proportional to the capacity to evolve new protein functions. Instead, structural features that are conserved among proteins with diverse functions might contribute to the evolution of new functions.

The protein databases contain many proteins with unknown function. A computational approach for predicting ligand specificity that requires only the sequence of the unknown protein would be valuable for directing experiment-based assignment of function. We focused on a family of unknown proteins in the mechanistically diverse enolase superfamily and used two approaches to assign function: (i) enzymatic assays using libraries of potential substrates, and (ii) in silico docking of the same libraries using a homology model based on the most similar (35% sequence identity) characterized protein. The results matched closely; an experimentally determined structure confirmed the predicted structure of the substrate-liganded complex. We assigned the N -succinyl arginine/lysine racemase function to the family, correcting the annotation ( L -Ala- D/L -Glu epimerase) based on the function of the most similar characterized homolog. These studies establish that ligand docking to a homology model can facilitate functional assignment of unknown proteins by restricting the identities of the possible substrates that must be experimentally tested.

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis. One-fourth of the global population is estimated to be infected with Mtb, accounting for ∼1.3 million deaths in 2017. As part of the immune response to Mtb infection, macrophages produce metabolites with the purpose of inhibiting or killing the bacterial cell. Itaconate is an abundant host metabolite thought to be both an antimicrobial agent and a modulator of the host inflammatory response. However, the exact mode of action of itaconate remains unclear. Here, we show that Mtb has an itaconate dissimilation pathway and that the last enzyme in this pathway, Rv2498c, also participates in L-leucine catabolism. Our results from phylogenetic analysis, in vitro enzymatic assays, X-ray crystallography, and in vivo Mtb experiments, identified Mtb Rv2498c as a bifunctional β-hydroxyacyl-CoA lyase and that deletion of the rv2498c gene from the Mtb genome resulted in attenuation in a mouse infection model. Altogether, this report describes an itaconate resistance mechanism in Mtb and an L-leucine catabolic pathway that proceeds via an unprecedented (R)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) stereospecific route in nature.

The vast number of poorly characterised enzymes in Mycobacterium tuberculosis (Mtb) is one of the key barriers precluding a better understanding of the biology that underpins pathogenesis. Here, we investigated the Mtb orphan enzyme Rv2498c to delineate its physiological role. Our results from in vitro enzymatic assays, phylogenetic analysis, X-ray crystallography and in vivo Mtb experiments, de-orphan Rv2498c as a multi-functional β-hydroxyacyl-CoA lyase/thioesterase (β-HAClyase/thioesterase) that participates in three different metabolic pathways: L-leucine catabolism, itaconate dissimilation, and glyoxylate shunt. Moreover, the deletion of the rv2498c gene from the Mtb genome resulted in attenuation in the mouse model compared to infection with the parent strain. To the best of our knowledge, this is the first report of an (R)-3-hydroxyl-3-methylglutaryl-CoA for leucine catabolism and an itaconate-specific resistance mechanism in Mtb.

The vast number of poorly characterised enzymes in Mycobacterium tuberculosis (Mtb) is one of the key barriers precluding a better understanding of the biology that underpins pathogenesis. Here, we investigated the Mtb orphan enzyme Rv2498c to delineate its physiological role. Our results from in vitro enzymatic assays, phylogenetic analysis, X-ray crystallography and in vivo Mtb experiments, de-orphan Rv2498c as a multi-functional -hydroxyacyl-CoA lyase/thioesterase ( -HAClyase/thioesterase) that participates in three different metabolic pathways: L-leucine catabolism, itaconate dissimilation, and glyoxylate shunt. Moreover, the deletion of the rv2498c gene from the Mtb genome resulted in attenuation in the mouse model compared to infection with the parent strain. To the best of our knowledge, this is the first report of an (R)-3-hydroxyl-3-methylglutaryl-CoA for leucine catabolism and an itaconate-specific resistance mechanism in Mtb.

Carbon dots (CDs) are light-emitting nanoparticles that show great promise for applications in biology and medicine due to the ease of fabrication, biocompatibility, and attractive optical properties. Optical chirality, on the other hand, is an intrinsic feature inherent in many objects in nature, and it can play an important role in the formation of artificial complexes based on CDs that are implemented for enantiomer recognition, site-specific bonding, etc. We employed a one-step hydrothermal synthesis to produce chiral CDs from the commonly used precursors citric acid and ethylenediamine together with a set of different chiral precursors, namely, L-isomers of cysteine, glutathione, phenylglycine, and tryptophan. The resulting CDs consisted of O,N-doped (and also S-doped, in some cases) carbonized cores with surfaces rich in amide and hydroxyl groups; they exhibited high photoluminescence quantum yields reaching 57%, chiral optical signals in the UV and visible spectral regions, and two-photon absorption. Chiral signals of CDs were rather complex and originated from a combination of the chiral precursors attached to the CD surface, hybridization of lower-energy levels of chiral chromophores formed within CDs, and intrinsic chirality of the CD cores. Using DFT analysis, we showed how incorporation of the chiral precursors at the optical centers induced a strong response in their circular dichroism spectra. The optical characteristics of these CDs, which can easily be dispersed in solvents of different polarities, remained stable during pH changes in the environment and after UV exposure for more than 400 min, which opens a wide range of bio-applications.Using different chiral precursor molecules, we produced chiral CDs with superior optical properties, such as two-photon absorption, high photoluminescence quantum yield reaching 57%, and chiral signals in the UV-Vis spectral region.

Carbon dots (CDs) are an attractive class of nanomaterials due to the ease of their synthesis, biocompatibility, and superior optical properties. The electronic structure of CDs and hence their optical transitions can be controlled and tuned over a wide spectral range via the choice of precursors, adjustment of the synthetic conditions, and post-synthetic treatment. We summarize recent progress in the synthesis of CDs emitting in different colors in terms of morphology and optical properties of the resulting nanoparticles, with a focus on the synthetic approaches allowing to shift their emission to longer wavelengths. We further consider formation of CD-based composite materials, and review approaches used to prevent aggregation and self-quenching of their emission. We then provide examples of applications of CDs in optoelectronic devices, such as solar cells and light-emitting diodes (LEDs) with a focus on white LEDs.

The possibilities of chlorophyll a (Chl a) and total suspended matter (TSM) retrieval using Sentinel-2/MSI imagery and in situ measurements in the Gorky Reservoir are investigated. This water body is an inland freshwater ecosystem within the territory of the Russian Federation. During the algal bloom period, the optical properties of water are extremely heterogeneous and vary on scales of tens of meters. Additionally, they vary in time under the influence of currents and wind forcing. In this case, the usage of the traditional station-based sampling to describe the state of the reservoir may be uninformative and not rational. Therefore, we proposed an original approach based on simultaneous in situ measurements of the remote sensing reflectance by a single radiometer and the concentration of water constituents by an ultraviolet fluorescence LiDAR from a high-speed gliding motorboat. This approach provided fast data collection including 4087 synchronized LiDAR and radiometric measurements with high spatial resolutions of 8 m for two hours. A part of the dataset was coincided with Sentinel-2 overpass and used for the development of regional algorithms for the retrieval of Chl a and TSM concentrations. For inland waters of the Russian Federation, such research was performed for the first time. The proposed algorithms can be used for regular environmental monitoring of the Gorky Reservoir using ship measurements or Sentinel-2 images. Additionally, they can be adapted for neighboring reservoirs, for example, for other seven reservoirs on the Volga River. Moreover, the proposed ship measurement approach can be useful in the practice of limnological monitoring of inland freshwater ecosystems with high spatiotemporal variability of the optical properties.