Interplay of halogen bonding and solvation in protein-ligand binding.

Halogen bonding is increasingly utilized in efforts to achieve high affinity and selectivity of molecules designed to bind proteins, making it paramount to understand the relationship between structure, dynamics, and thermodynamic driving forces. We present a detailed analysis addressing this problem using a series of protein-ligand complexes involving single halogen substitutions - F, Cl, Br, and I - and nearly identical structures. Isothermal titration calorimetry reveals an increasingly favorable binding enthalpy from F to I that correlates with the halogen size and σ-hole electropositive character, but is partially counteracted by unfavorable entropy, which is constant from F to Cl and Br, but worse for I. Consequently, the binding free energy is roughly equal for Cl, Br, and I. QM and solvation-free-energy calculations reflect an intricate balance between halogen bonding, hydrogen bonds, and solvation. These advances have the potential to aid future drug design initiatives involving halogenated compounds.

Ligand Sulfur Oxidation State Progressively Alters Galectin-3-Ligand Complex Conformations To Induce Affinity-Influencing Hydrogen Bonds.

Galectins play biological roles in immune regulation and tumor progression. Ligands with high affinity for the shallow, hydrophilic galectin-3 ligand binding site rely primarily on a galactose core with appended aryltriazole moieties, making hydrophobic interactions and π-stacking. We designed and synthesized phenyl sulfone, sulfoxide, and sulfide-triazolyl thiogalactoside derivatives to create affinity-enhancing hydrogen bonds, hydrophobic and π-interactions. Crystal structures and thermodynamic analyses revealed that the sulfoxide and sulfone ligands form hydrogen bonds while retaining π-interactions, resulting in improved affinities and unique binding poses. The sulfoxide, bearing one hydrogen bond acceptor, leads to an affinity decrease compared to the sulfide, whereas the corresponding sulfone forms three hydrogen bonds, two directly with Asn and Arg side chains and one water-mediated to an Asp side chain, respectively, which alters the complex structure and increases affinity. These findings highlight that the sulfur oxidation state influences both the interaction thermodynamics and structure.

Structural basis of activation and antagonism of receptor signaling mediated by interleukin-27.

Interleukin-27 (IL-27) uniquely assembles p28 and EBI3 subunits to a heterodimeric cytokine that signals via IL-27Rα and gp130. To provide the structural framework for receptor activation by IL-27 and its emerging therapeutic targeting, we report here crystal structures of mouse IL-27 in complex with IL-27Rα and of human IL-27 in complex with SRF388, a monoclonal antibody undergoing clinical trials with oncology indications. One face of the helical p28 subunit interacts with EBI3, while the opposite face nestles into the interdomain elbow of IL-27Rα to juxtapose IL-27Rα to EBI3. This orients IL-27Rα for paired signaling with gp130, which only uses its immunoglobulin domain to bind to IL-27. Such a signaling complex is distinct from those mediated by IL-12 and IL-23. The SRF388 binding epitope on IL-27 overlaps with the IL-27Rα interaction site explaining its potent antagonistic properties. Collectively, our findings will facilitate the mechanistic interrogation, engineering, and therapeutic targeting of IL-27.

Dusquetide modulates innate immune response through binding to p62.

SQSTM1/p62 is an autophagic receptor that plays a major role in mediating stress and innate immune responses. Preclinical studies identified p62 as a target of the prototype innate defense regulator (IDR); however, the molecular mechanism of this process remains unclear. Here, we describe the structural basis and biological consequences of the interaction of p62 with the next generation of IDRs, dusquetide. Both electrostatic and hydrophobic contacts drive the formation of the complex between dusquetide and the ZZ domain of p62. We show that dusquetide penetrates the cell membrane and associates with p62 in vivo. Dusquetide binding modulates the p62-RIP1 complex, increases p38 phosphorylation, and enhances CEBP/B expression without activating autophagy. Our findings provide molecular details underlying the IDR action that may help in the development of new strategies to pharmacologically target p62.

A nucleotide-sensing oligomerization mechanism that controls NrdR-dependent transcription of ribonucleotide reductases.

Ribonucleotide reductase (RNR) is an essential enzyme that catalyzes the synthesis of DNA building blocks in virtually all living cells. NrdR, an RNR-specific repressor, controls the transcription of RNR genes and, often, its own, in most bacteria and some archaea. NrdR senses the concentration of nucleotides through its ATP-cone, an evolutionarily mobile domain that also regulates the enzymatic activity of many RNRs, while a Zn-ribbon domain mediates binding to NrdR boxes upstream of and overlapping the transcription start site of RNR genes. Here, we combine biochemical and cryo-EM studies of NrdR from Streptomyces coelicolor to show, at atomic resolution, how NrdR binds to DNA. The suggested mechanism involves an initial dodecamer loaded with two ATP molecules that cannot bind to DNA. When dATP concentrations increase, an octamer forms that is loaded with one molecule each of dATP and ATP per monomer. A tetramer derived from this octamer then binds to DNA and represses transcription of RNR. In many bacteria - including well-known pathogens such as Mycobacterium tuberculosis - NrdR simultaneously controls multiple RNRs and hence DNA synthesis, making it an excellent target for novel antibiotics development.

The engineered CD80 variant fusion therapeutic davoceticept combines checkpoint antagonism with conditional CD28 costimulation for anti-tumor immunity.

Despite the recent clinical success of T cell checkpoint inhibition targeting the CTLA-4 and PD-1 pathways, many patients either fail to achieve objective responses or they develop resistance to therapy. In some cases, poor responses to checkpoint blockade have been linked to suboptimal CD28 costimulation and the inability to generate and maintain a productive adaptive anti-tumor immune response. To address this, here we utilize directed evolution to engineer a CD80 IgV domain with increased PD-L1 affinity and fuse this to an immunoglobulin Fc domain, creating a therapeutic (ALPN-202, davoceticept) capable of providing CD28 costimulation in a PD-L1-dependent fashion while also antagonizing PD-1 - PD-L1 and CTLA-4-CD80/CD86 interactions. We demonstrate that by combining CD28 costimulation and dual checkpoint inhibition, ALPN-202 enhances T cell activation and anti-tumor efficacy in cell-based assays and mouse tumor models more potently than checkpoint blockade alone and thus has the potential to generate potent, clinically meaningful anti-tumor immunity in humans.

Structures of activin ligand traps using natural sets of type I and type II TGFß receptors.

The 30+ unique ligands of the TGFß family signal by forming complexes using different combinations of type I and type II receptors. Therapeutically, the extracellular domain of a single receptor fused to an Fc molecule can effectively neutralize subsets of ligands. Increased ligand specificity can be accomplished by using the extracellular domains of both the type I and type II receptor to mimic the naturally occurring signaling complex. Here, we report the structure of one "type II-type I-Fc" fusion, ActRIIB-Alk4-Fc, in complex with two TGFß family ligands, ActA, and GDF11, providing a snapshot of this therapeutic platform. The study reveals that extensive contacts are formed by both receptors, replicating the ternary signaling complex, despite the inherent low affinity of Alk4. Our study shows that low-affinity type I interactions support altered ligand specificity and can be visualized at the molecular level using this platform.

Entropy-Entropy Compensation between the Protein, Ligand, and Solvent Degrees of Freedom Fine-Tunes Affinity in Ligand Binding to Galectin-3C.

Molecular recognition is fundamental to biological signaling. A central question is how individual interactions between molecular moieties affect the thermodynamics of ligand binding to proteins and how these effects might propagate beyond the immediate neighborhood of the binding site. Here, we investigate this question by introducing minor changes in ligand structure and characterizing the effects of these on ligand affinity to the carbohydrate recognition domain of galectin-3, using a combination of isothermal titration calorimetry, X-ray crystallography, NMR relaxation, and computational approaches including molecular dynamics (MD) simulations and grid inhomogeneous solvation theory (GIST). We studied a congeneric series of ligands with a fluorophenyl-triazole moiety, where the fluorine substituent varies between the ortho, meta, and para positions (denoted O, M, and P). The M and P ligands have similar affinities, whereas the O ligand has 3-fold lower affinity, reflecting differences in binding enthalpy and entropy. The results reveal surprising differences in conformational and solvation entropy among the three complexes. NMR backbone order parameters show that the O-bound protein has reduced conformational entropy compared to the M and P complexes. By contrast, the bound ligand is more flexible in the O complex, as determined by 19F NMR relaxation, ensemble-refined X-ray diffraction data, and MD simulations. Furthermore, GIST calculations indicate that the O-bound complex has less unfavorable solvation entropy compared to the other two complexes. Thus, the results indicate compensatory effects from ligand conformational entropy and water entropy, on the one hand, and protein conformational entropy, on the other hand. Taken together, these different contributions amount to entropy-entropy compensation among the system components involved in ligand binding to a target protein.

NAD+ pool depletion as a signal for the Rex regulon involved in Streptococcus agalactiae virulence.

In many Gram-positive bacteria, the redox-sensing transcriptional repressor Rex controls central carbon and energy metabolism by sensing the intra cellular balance between the reduced and oxidized forms of nicotinamide adenine dinucleotide; the NADH/NAD+ ratio. Here, we report high-resolution crystal structures and characterization of a Rex ortholog (Gbs1167) in the opportunistic pathogen, Streptococcus agalactiae, also known as group B streptococcus (GBS). We present structures of Rex bound to NAD+ and to a DNA operator which are the first structures of a Rex-family member from a pathogenic bacterium. The structures reveal the molecular basis of DNA binding and the conformation alterations between the free NAD+ complex and DNA-bound form of Rex. Transcriptomic analysis revealed that GBS Rex controls not only central metabolism, but also expression of the monocistronic rex gene as well as virulence gene expression. Rex enhances GBS virulence after disseminated infection in mice. Mechanistically, NAD+ stabilizes Rex as a repressor in the absence of NADH. However, GBS Rex is unique compared to Rex regulators previously characterized because of its sensing mechanism: we show that it primarily responds to NAD+ levels (or growth rate) rather than to the NADH/NAD+ ratio. These results indicate that Rex plays a key role in GBS pathogenicity by modulating virulence factor gene expression and carbon metabolism to harvest nutrients from the host.

Exploring ligand dynamics in protein crystal structures with ensemble refinement.

Understanding the dynamics of ligands bound to proteins is an important task in medicinal chemistry and drug design. However, the dominant technique for determining protein-ligand structures, X-ray crystallography, does not fully account for dynamics and cannot accurately describe the movements of ligands in protein binding sites. In this article, an alternative method, ensemble refinement, is used on six protein-ligand complexes with the aim of understanding the conformational diversity of ligands in protein crystal structures. The results show that ensemble refinement sometimes indicates that the flexibility of parts of the ligand and some protein side chains is larger than that which can be described by a single conformation and atomic displacement parameters. However, since the electron-density maps are comparable and Rfree values are slightly increased, the original crystal structure is still a better model from a statistical point of view. On the other hand, it is shown that molecular-dynamics simulations and automatic generation of alternative conformations in crystallographic refinement confirm that the flexibility of these groups is larger than is observed in standard refinement. Moreover, the flexible groups in ensemble refinement coincide with groups that give high atomic displacement parameters or non-unity occupancy if optimized in standard refinement. Therefore, the conformational diversity indicated by ensemble refinement seems to be qualitatively correct, indicating that ensemble refinement can be an important complement to standard crystallographic refinement as a tool to discover which parts of crystal structures may show extensive flexibility and therefore are poorly described by a single conformation. However, the diversity of the ensembles is often exaggerated (probably partly owing to the rather poor force field employed) and the ensembles should not be trusted in detail.

Solution Structure of the dATP-Inactivated Class I Ribonucleotide Reductase From Leeuwenhoekiella blandensis by SAXS and Cryo-Electron Microscopy.

The essential enzyme ribonucleotide reductase (RNR) is highly regulated both at the level of overall activity and substrate specificity. Studies of class I, aerobic RNRs have shown that overall activity is downregulated by the binding of dATP to a small domain known as the ATP-cone often found at the N-terminus of RNR subunits, causing oligomerization that prevents formation of a necessary α2ß2 complex between the catalytic (α2) and radical generating (ß2) subunits. In some relatively rare organisms with RNRs of the subclass NrdAi, the ATP-cone is found at the N-terminus of the ß subunit rather than more commonly the α subunit. Binding of dATP to the ATP-cone in ß results in formation of an unusual ß4 tetramer. However, the structural basis for how the formation of the active complex is hindered by such oligomerization has not been studied. Here we analyse the low-resolution three-dimensional structures of the separate subunits of an RNR from subclass NrdAi, as well as the α4ß4 octamer that forms in the presence of dATP. The results reveal a type of oligomer not previously seen for any class of RNR and suggest a mechanism for how binding of dATP to the ATP-cone switches off catalysis by sterically preventing formation of the asymmetrical α2ß2 complex.

DutaFabs are engineered therapeutic Fab fragments that can bind two targets simultaneously.

We report the development of a platform of dual targeting Fab (DutaFab) molecules, which comprise two spatially separated and independent binding sites within the human antibody CDR loops: the so-called H-side paratope encompassing HCDR1, HCDR3 and LCDR2, and the L-side paratope encompassing LCDR1, LCDR3 and HCDR2. Both paratopes can be independently selected and combined into the desired bispecific DutaFabs in a modular manner. X-ray crystal structures illustrate that DutaFabs are able to bind two target molecules simultaneously at the same Fv region comprising a VH-VL heterodimer. In the present study, this platform is applied to generate DutaFabs specific for VEGFA and PDGF-BB, which show high affinities, physico-chemical stability and solubility, as well as superior efficacy over anti-VEGF monotherapy in vivo. These molecules exemplify the usefulness of DutaFabs as a distinct class of antibody therapeutics, which is currently being evaluated in patients.

Exposing the distinctive modular behavior of ß-strands and α-helices in folded proteins.

Although folded proteins are commonly depicted as simplistic combinations of ß-strands and α-helices, the actual properties and functions of these secondary-structure elements in their native contexts are just partly understood. The principal reason is that the behavior of individual ß- and α-elements is obscured by the global folding cooperativity. In this study, we have circumvented this problem by designing frustrated variants of the mixed α/ß-protein S6, which allow the structural behavior of individual ß-strands and α-helices to be targeted selectively by stopped-flow kinetics, X-ray crystallography, and solution-state NMR. Essentially, our approach is based on provoking intramolecular "domain swap." The results show that the α- and ß-elements have quite different characteristics: The swaps of ß-strands proceed via global unfolding, whereas the α-helices are free to swap locally in the native basin. Moreover, the α-helices tend to hybridize and to promote protein association by gliding over to neighboring molecules. This difference in structural behavior follows directly from hydrogen-bonding restrictions and suggests that the protein secondary structure defines not only tertiary geometry, but also maintains control in function and structural evolution. Finally, our alternative approach to protein folding and native-state dynamics presents a generally applicable strategy for in silico design of protein models that are computationally testable in the microsecond-millisecond regime.

Current status and future opportunities for serial crystallography at MAX IV Laboratory.

Over the last decade, serial crystallography, a method to collect complete diffraction datasets from a large number of microcrystals delivered and exposed to an X-ray beam in random orientations at room temperature, has been successfully implemented at X-ray free-electron lasers and synchrotron radiation facility beamlines. This development relies on a growing variety of sample presentation methods, including different fixed target supports, injection methods using gas-dynamic virtual-nozzle injectors and high-viscosity extrusion injectors, and acoustic levitation of droplets, each with unique requirements. In comparison with X-ray free-electron lasers, increased beam time availability makes synchrotron facilities very attractive to perform serial synchrotron X-ray crystallography (SSX) experiments. Within this work, the possibilities to perform SSX at BioMAX, the first macromolecular crystallography beamline at  MAX IV Laboratory in Lund, Sweden, are described, together with case studies from the SSX user program: an implementation of a high-viscosity extrusion injector to perform room temperature serial crystallography at BioMAX using two solid supports - silicon nitride membranes (Silson, UK) and XtalTool (Jena Bioscience, Germany). Future perspectives for the dedicated serial crystallography beamline MicroMAX at MAX IV Laboratory, which will provide parallel and intense micrometre-sized X-ray beams, are discussed.

FragMAX: the fragment-screening platform at the MAX IV Laboratory.

Advances in synchrotron storage rings and beamline automation have pushed data-collection rates to thousands of data sets per week. With this increase in throughput, massive projects such as in-crystal fragment screening have become accessible to a larger number of research groups. The quality of support offered at large-scale facilities allows medicinal chemistry-focused or biochemistry-focused groups to supplement their research with structural biology. Preparing the experiment, analysing multiple data sets and prospecting for interesting complexes of protein and fragments require, for both newcomers and experienced users, efficient management of the project and extensive computational power for data processing and structure refinement. Here, FragMAX, a new complete platform for fragment screening at the BioMAX beamline of the MAX IV Laboratory, is described. The ways in which users are assisted in X-ray-based fragment screenings and in which the fourth-generation storage ring available at the facility is best exploited are also described.

Interactive model building in neutron macromolecular crystallography.

This chapter aims to give an overview of the process of interactive model building in macromolecular neutron crystallography for the researcher transitioning from X-ray crystallography alone. The two most popular programs for refinement and model building, phenix.refine and Coot, respectively, are used as examples, and familiarity with the programs is assumed. Some work-arounds currently required for proper communication between the programs are described. We also discuss the appearance of nuclear density maps and how this differs from that of electron density maps. Advice is given to facilitate deposition of jointly refined neutron/X-ray structures in the Protein Data Bank.

Refinement of protein structures using a combination of quantum-mechanical calculations with neutron and X-ray crystallographic data. Corrigendum.

Corrections are published for the article by Caldararu et al. [(2019), Acta Cryst. D75, 368-380].

3-Substituted 1-Naphthamidomethyl-C-galactosyls Interact with Two Unique Sub-sites for High-Affinity and High-Selectivity Inhibition of Galectin-3.

The galectins are a family of galactose-binding proteins playing key roles in inflammatory processes and cancer. However, they are structurally very closely related, and discovery of highly selective inhibitors is challenging. In this work, we report the design of novel inhibitors binding to a subsite unique to galectin-3, which confers both high selectivity and affinity towards galectin-3. Olefin cross metathesis between allyl ß-C-galactopyranosyl and 1-vinylnaphthalenes or acylation of aminomethyl ß-C-galactopyranosyl with 1-naphthoic acid derivatives gave C-galactopyranosyls carrying 1-naphthamide structural elements that interacted favorably with a galectin-3 unique subsite according to molecular modeling and X-ray structural analysis of two inhibitor-galectin-3 complexes. Affinities were down to sub-µM and selectivities over galectin-1, 2, 4 N-terminal domain, 4 C-terminal domain, 7, 8 N-terminal domain, 9 N-terminal domain, and 9 C-terminal domain were high. These results show that high affinity and selectivity for a single galectin can be achieved by targeting unique subsites, which holds promise for further development of small and selective galectin inhibitors.

Insight into the dimer dissociation process of the Chromobacterium violaceum (S)-selective amine transaminase.

One of the main factors hampering the implementation in industry of transaminase-based processes for the synthesis of enantiopure amines is their often low storage and operational stability. Our still limited understanding of the inactivation processes undermining the stability of wild-type transaminases represents an obstacle to improving their stability through enzyme engineering. In this paper we present a model describing the inactivation process of the well-characterized (S)-selective amine transaminase from Chromobacterium violaceum. The cornerstone of the model, supported by structural, computational, mutagenesis and biophysical data, is the central role of the catalytic lysine as a conformational switch. Upon breakage of the lysine-PLP Schiff base, the strain associated with the catalytically active lysine conformation is dissipated in a slow relaxation process capable of triggering the known structural rearrangements occurring in the holo-to-apo transition and ultimately promoting dimer dissociation. Due to the occurrence in the literature of similar PLP-dependent inactivation models valid for other non-transaminase enzymes belonging to the same fold-class, the role of the catalytic lysine as conformational switch might extend beyond the transaminase enzyme group and offer new insight to drive future non-trivial engineering strategies.

Are crystallographic B-factors suitable for calculating protein conformational entropy?

Conformational entropies are of great interest when studying the binding of small ligands to proteins or the interaction of proteins. Unfortunately, there are no experimental methods available to measure conformational entropies of all groups in a protein. Instead, they are normally estimated from molecular dynamics (MD) simulations, although such methods show problems with convergence and correlation of motions, and depend on the accuracy of the underlying potential-energy function. Crystallographic atomic displacement parameters (also known as B-factors) are available in all crystal structures and contain information about the atomic fluctuations, which can be converted to entropies. We have studied whether B-factors can be employed to extract conformational entropies for proteins by comparing such entropies to those measured by NMR relaxation experiments or obtained from MD simulations in solution or in the crystal. Unfortunately, our results show that B-factor entropies are unreliable, because they include the movement and rotation of the entire protein, they exclude correlation of the movements and they include contributions other than the fluctuations, e.g. static disorder, as well as errors in the model and the scattering factors. We have tried to reduce the first problem by employing translation-libration-screw refinement, the second by employing a description of the correlated movement from MD simulations, and the third by studying only the change in entropy when a pair of ligands binds to the same protein, thoroughly re-refining the structures in exactly the same way and using the same set of alternative conformations. However, the experimental B-factors seem to be incompatible with fluctuations from MD simulations and the precision is too poor to give any reliable entropies.