Improving Protein Expression, Stability, and Function with ProteinMPNN.

Natural proteins are highly optimized for function but are often difficult to produce at a scale suitable for biotechnological applications due to poor expression in heterologous systems, limited solubility, and sensitivity to temperature. Thus, a general method that improves the physical properties of native proteins while maintaining function could have wide utility for protein-based technologies. Here, we show that the deep neural network ProteinMPNN, together with evolutionary and structural information, provides a route to increasing protein expression, stability, and function. For both myoglobin and tobacco etch virus (TEV) protease, we generated designs with improved expression, elevated melting temperatures, and improved function. For TEV protease, we identified multiple designs with improved catalytic activity as compared to the parent sequence and previously reported TEV variants. Our approach should be broadly useful for improving the expression, stability, and function of biotechnologically important proteins.

Expanding the Substrate Scope of Acyltransferase LovD9 for the Biosynthesis of Statin Analogues.

Invited for the cover of this issue are the groups of Gonzalo Jiménez-Osés and Fernando López-Gallego at CIC bioGUNE and CIC biomaGUNE, respectively. The image depicts the substrate scope of an engineered acyl transferases for the synthesis of statin derivatives. Read the full text of the article at 10.1002/chem.202300911.

Expanding the Substrate Scope of Acyltransferase LovD9 for the Biosynthesis of Statin Analogues.

This study identifies new acyl donors for manufacturing statin analogues through the acylation of monacolin J acid by the laboratory evolved acyltransferase LovD9. Vinyl and p-nitrophenyl esters have emerged as alternate substrates for LovD9-catalyzed acylation. While vinyl esters can reach product yields as high as the ones obtained by α-dimethyl butyryl-S-methyl-3-mercaptopropionate (DMB-SMMP), the thioester for which LovD9 was evolved, p-nitrophenyl esters display a reactivity even higher than DMB-SMMP for the first acylation step yet the acylation product yield is lower. The reaction mechanisms were elucidated through quantum mechanics (QM) calculations.

Structures of the Inhibitory Receptor Siglec-8 in Complex with a High-Affinity Sialoside Analogue and a Therapeutic Antibody.

Human sialic acid binding immunoglobulin-like lectin-8 (Siglec-8) is an inhibitory receptor that triggers eosinophil apoptosis and can inhibit mast cell degranulation when engaged by specific monoclonal antibodies (mAbs) or sialylated ligands. Thus, Siglec-8 has emerged as a critical negative regulator of inflammatory responses in diverse diseases, such as allergic airway inflammation. Herein, we have deciphered the molecular recognition features of the interaction of Siglec-8 with the mAb lirentelimab (2C4, under clinical development) and with a sialoside mimetic with the potential to suppress mast cell degranulation. The three-dimensional structure of Siglec-8 and the fragment antigen binding (Fab) portion of the anti-Siglec-8 mAb 2C4, solved by X-ray crystallography, reveal that 2C4 binds close to the carbohydrate recognition domain (V-type Ig domain) on Siglec-8. We have also deduced the binding mode of a high-affinity analogue of its sialic acid ligand (9-N-napthylsufonimide-Neu5Ac, NSANeuAc) using a combination of NMR spectroscopy and X-ray crystallography. Our results show that the sialoside ring of NSANeuAc binds to the canonical sialyl binding pocket of the Siglec receptor family and that the high affinity arises from the accommodation of the NSA aromatic group in a nearby hydrophobic patch formed by the N-terminal tail and the unique G-G' loop. The results reveal the basis for the observed high affinity of this ligand and provide clues for the rational design of the next generation of Siglec-8 inhibitors. Additionally, the specific interactions between Siglec-8 and the N-linked glycans present on the high-affinity receptor FcεRIα have also been explored by NMR.

Distal Mutations Shape Substrate-Binding Sites during Evolution of a Metallo-Oxidase into a Laccase.

Laccases are in increasing demand as innovative solutions in the biorefinery fields. Here, we combine mutagenesis with structural, kinetic, and in silico analyses to characterize the molecular features that cause the evolution of a hyperthermostable metallo-oxidase from the multicopper oxidase family into a laccase (k cat 273 s-1 for a bulky aromatic substrate). We show that six mutations scattered across the enzyme collectively modulate dynamics to improve the binding and catalysis of a bulky aromatic substrate. The replacement of residues during the early stages of evolution is a stepping stone for altering the shape and size of substrate-binding sites. Binding sites are then fine-tuned through high-order epistasis interactions by inserting distal mutations during later stages of evolution. Allosterically coupled, long-range dynamic networks favor catalytically competent conformational states that are more suitable for recognizing and stabilizing the aromatic substrate. This work provides mechanistic insight into enzymatic and evolutionary molecular mechanisms and spots the importance of iterative experimental and computational analyses to understand local-to-global changes.

Unveiling the role of pyrylium frameworks on π-stacking interactions: a combined ab initio and experimental study.

A multidisciplinary study is presented to shed light on how pyrylium frameworks, as π-hole donors, establish π-π interactions. The combination of CSD analysis, computational modelling (ab intitio, DFT and MD simulations) and experimental NMR spectroscopy data provides essential information on the key parameters that characterize these intereactions, opening new avenues for further applications of this versatile heterocycle.

A Computational Perspective on Molecular Recognition by Galectins.

This article presents an overview of recent computational studies dedicated to the analysis of binding between galectins and small-molecule ligands. We first present a summary of the most popular simulation techniques adopted for calculating binding poses and binding energies and then discuss relevant examples reported in the literature for the three main classes of galectins (dimeric, tandem, and chimera). We show that simulation of galectin-ligand interactions is a mature field that has proven invaluable for completing and unraveling experimental observations. Future perspectives to further improve the accuracy and cost-effectiveness of existing computational approaches will involve the development of new schemes to account for solvation and entropy effects, which represent the main current limitations to the accuracy of computational results.

Structural insight into the unique conformation of cystathionine ß-synthase from Toxoplasma gondii.

Cysteine plays a major role in the redox homeostasis and antioxidative defense mechanisms of many parasites of the phylum Apicomplexa. Of relevance to human health is Toxoplasma gondii, the causative agent of toxoplasmosis. A major route of cysteine biosynthesis in this parasite is the reverse transsulfuration pathway involving two key enzymes cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CGL). CBS from T. gondii (TgCBS) catalyzes the pyridoxal-5́-phosphate-dependent condensation of homocysteine with either serine or O-acetylserine to produce cystathionine. The enzyme can perform alternative reactions that use homocysteine and cysteine as substrates leading to the endogenous biosynthesis of hydrogen sulfide, another key element in maintaining the intracellular redox equilibrium. In contrast with human CBS, TgCBS lacks the N-terminal heme binding domain and is not responsive to S-adenosylmethionine. Herein, we describe the structure of a TgCBS construct that lacks amino acid residues 466-491 and shows the same activity of the native protein. TgCBS Δ466-491 was determined alone and in complex with reaction intermediates. A complementary molecular dynamics analysis revealed a unique domain organization, similar to the pathogenic mutant D444N of human CBS. Our data provides one missing piece in the structural diversity of CBSs by revealing the so far unknown three-dimensional arrangement of the CBS-type of Apicomplexa. This domain distribution is also detected in yeast and bacteria like Pseudomonas aeruginosa. These results pave the way for understanding the mechanisms by which TgCBS regulates the intracellular redox of the parasite, and have far-reaching consequences for the functional understanding of CBSs with similar domain distribution.

The two domains of human galectin-8 bind sialyl- and fucose-containing oligosaccharides in an independent manner. A 3D view by using NMR.

The interaction of human galectin-8 and its two separate N-terminal and C-terminal carbohydrate recognition domains (CRD) to their natural ligands has been analysed using a synergistic combination of experimental NMR and ITC methods, and molecular dynamics simulations. Both domains bind the minimal epitopes N-acetyllactosamine (1) and Galß1-3GalNAc (2) in a similar manner. However, the N-terminal and C-terminal domains show exquisite and opposing specificity to bind either Neu5Ac- or Fuc-containing ligands, respectively. Moreover, the addition of the high-affinity ligands specific for one of the CRDs does not make any effect on the binding at the alternative one. Thus, the two CRDs behave independently and may simultaneously target different molecular entities to promote clustering through the generation of supramolecular assemblies.

Galectin-4 N-Terminal Domain: Binding Preferences Toward A and B Antigens With Different Peripheral Core Presentations.

The tandem-repeat Galectin-4 (Gal-4) contains two different domains covalently linked through a short flexible peptide. Both domains have been shown to bind preferentially to A and B histo blood group antigens with different affinities, although the binding details are not yet available. The biological relevance of these associations is unknown, although it could be related to its attributed role in pathogen recognition. The presentation of A and B histo blood group antigens in terms of peripheral core structures differs among tissues and from that of the antigen-mimicking structures produced by pathogens. Herein, the binding of the N-terminal domain of Gal-4 toward a group of differently presented A and B oligosaccharide antigens in solution has been studied through a combination of NMR, isothermal titration calorimetry (ITC), and molecular modeling. The data presented in this paper allow the identification of the specific effects that subtle chemical modifications within this antigenic family have in the binding to the N-terminal domain of Gal-4 in terms of affinity and intermolecular interactions, providing a structural-based rationale for the observed trend in the binding preferences.

Unravelling the Time Scale of Conformational Plasticity and Allostery in Glycan Recognition by Human Galectin-1.

The interaction of human galectin-1 with a variety of oligosaccharides, from di-(N-acetyllactosamine) to tetra-saccharides (blood B type-II antigen) has been scrutinized by using a combined approach of different NMR experiments, molecular dynamics (MD) simulations, and isothermal titration calorimetry. Ligand- and receptor-based NMR experiments assisted by computational methods allowed proposing three-dimensional structures for the different complexes, which explained the lack of enthalpy gain when increasing the chemical complexity of the glycan. Interestingly, and independently of the glycan ligand, the entropy term does not oppose the binding event, a rather unusual feature for protein-sugar interactions. CLEANEX-PM and relaxation dispersion experiments revealed that sugar binding affected residues far from the binding site and described significant changes in the dynamics of the protein. In particular, motions in the microsecond-millisecond timescale in residues at the protein dimer interface were identified in the presence of high affinity ligands. The dynamic process was further explored by extensive MD simulations, which provided additional support for the existence of allostery in glycan recognition by human galectin-1.

Precise Installation of Diazo-Tagged Side-Chains on Proteins to Enable In Vitro and In-Cell Site-Specific Labeling.

The chemistry of diazo compounds has generated a huge breadth of applications in the field of organic synthesis. Their versatility combined with their tunable reactivity, stability, and chemoselectivity makes diazo compounds desirable reagents for chemical biologists. Here, we describe a method for the precise installation of diazo handles on proteins and antibodies in a mild and specific approach. Subsequent 1,3-cycloaddition reactions with strained alkynes enable both bioimaging through an in-cell "click" reaction and probing of the cysteine proteome in cell lysates. The selectivity and efficiency of these processes makes these suitable reagents for chemical biology studies.

Fingerprinting the Nature of Anions in Pyrylium Complexes: Dual Binding Mode for Anion-π Interactions.

The interplay between noncovalent interactions that involve oxygenated heteroaromatic rings have been studied for the first time in this work. In particular, we report an advance in knowledge-based anion-π interactions together with (C-H)+ ⋅⋅⋅anion contacts. To understand how the anion modulates these interactions, the synthesis of pyrylium salts with a variety of anions was performed by using an anionic metathesis methodology. The synthesized pyrylium complexes were classified in series, for example, anions derived from halogens, from oxoacids, from p-block elements, and from transition metals. Crystallographic data, DFT calculations, and NMR spectroscopy methods provided access to an overall insight into the noncovalent behavior of the anion in this kind of system. Based on the DFT calculations and 1 H NMR spectroscopy, pyrylium protons can be used as chemical tags to detect noncovalent interactions in this type of compound.