Evolutionary conservation of the structure and function of meiotic Rec114-Mei4 and Mer2 complexes.

Meiosis-specific Rec114-Mei4 and Mer2 complexes are thought to enable Spo11-mediated DNA double-strand break (DSB) formation through a mechanism that involves DNA-dependent condensation. However, the structure, molecular properties, and evolutionary conservation of Rec114-Mei4 and Mer2 are unclear. Here, we present AlphaFold models of Rec114-Mei4 and Mer2 complexes supported by nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), and mutagenesis. We show that dimers composed of the Rec114 C terminus form α-helical chains that cup an N-terminal Mei4 α helix, and that Mer2 forms a parallel homotetrameric coiled coil. Both Rec114-Mei4 and Mer2 bind preferentially to branched DNA substrates, indicative of multivalent protein-DNA interactions. Indeed, the Rec114-Mei4 interaction domain contains two DNA-binding sites that point in opposite directions and drive condensation. The Mer2 coiled-coil domain bridges coaligned DNA duplexes, likely through extensive electrostatic interactions along the length of the coiled coil. Finally, we show that the structures of Rec114-Mei4 and Mer2 are conserved across eukaryotes, while DNA-binding properties vary significantly. This work provides insights into the mechanism whereby Rec114-Mei4 and Mer2 complexes promote the assembly of the meiotic DSB machinery and suggests a model in which Mer2 condensation is the essential driver of assembly, with the DNA-binding activity of Rec114-Mei4 playing a supportive role.

NMR-guided directed evolution.

Directed evolution is a powerful tool for improving existing properties and imparting completely new functionalities to proteins1-4. Nonetheless, its potential in even small proteins is inherently limited by the astronomical number of possible amino acid sequences. Sampling the complete sequence space of a 100-residue protein would require testing of 20100 combinations, which is beyond any existing experimental approach. In practice, selective modification of relatively few residues is sufficient for efficient improvement, functional enhancement and repurposing of existing proteins5. Moreover, computational methods have been developed to predict the locations and, in certain cases, identities of potentially productive mutations6-9. Importantly, all current approaches for prediction of hot spots and productive mutations rely heavily on structural information and/or bioinformatics, which is not always available for proteins of interest. Moreover, they offer a limited ability to identify beneficial mutations far from the active site, even though such changes may markedly improve the catalytic properties of an enzyme10. Machine learning methods have recently showed promise in predicting productive mutations11, but they frequently require large, high-quality training datasets, which are difficult to obtain in directed evolution experiments. Here we show that mutagenic hot spots in enzymes can be identified using NMR spectroscopy. In a proof-of-concept study, we converted myoglobin, a non-enzymatic oxygen storage protein, into a highly efficient Kemp eliminase using only three mutations. The observed levels of catalytic efficiency exceed those of proteins designed using current approaches and are similar with those of natural enzymes for the reactions that they are evolved to catalyse. Given the simplicity of this experimental approach, which requires no a priori structural or bioinformatic knowledge, we expect it to be widely applicable and to enable the full potential of directed enzyme evolution.

Beyond the VSG layer: Exploring the role of intrinsic disorder in the invariant surface glycoproteins of African trypanosomes.

In the bloodstream of mammalian hosts, African trypanosomes face the challenge of protecting their invariant surface receptors from immune detection. This crucial role is fulfilled by a dense, glycosylated protein layer composed of variant surface glycoproteins (VSGs), which undergo antigenic variation and provide a physical barrier that shields the underlying invariant surface glycoproteins (ISGs). The protective shield's limited permeability comes at the cost of restricted access to the extracellular host environment, raising questions regarding the specific function of the ISG repertoire. In this study, we employ an integrative structural biology approach to show that intrinsically disordered membrane-proximal regions are a common feature of members of the ISG super-family, conferring the ability to switch between compact and elongated conformers. While the folded, membrane-distal ectodomain is buried within the VSG layer for compact conformers, their elongated counterparts would enable the extension beyond it. This dynamic behavior enables ISGs to maintain a low immunogenic footprint while still allowing them to engage with the host environment when necessary. Our findings add further evidence to a dynamic molecular organization of trypanosome surface antigens wherein intrinsic disorder underpins the characteristics of a highly flexible ISG proteome to circumvent the constraints imposed by the VSG coat.

Structural insights into the interaction between adenovirus C5 hexon and human lactoferrin.

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.

Enantioselective, BroÌ·nsted Acid-Catalyzed Anti-selective Hydroamination of Alkenes.

Chiral pyrrolidines are common structural motives in natural products as well as active pharmaceutical ingredients, explaining the need for methods for their enantioselective synthesis. While several, often metal-catalyzed, methods for their preparation do exist, the enantioselective synthesis of pyrrolidines containing quaternary stereocenters remains challenging. Herein, we report a BroÌ·nsted acid-catalyzed intramolecular hydroamination that provides such pyrrolidines from simple starting materials in high yield and enantioselectivity. Key to an efficient reaction was the use of an electron-deficient protective group on nitrogen, the common nosyl-protecting group, to avoid deactivation of the BroÌ·nsted acid by deprotonation. The reaction proceeds as a stereospecific anti-addition indicating a concerted reaction. Furthermore, kinetic studies show Michaelis-Menten behavior, suggesting the formation of a precomplex similar to those observed in enzymatic catalysis.

Novel anti-repression mechanism of H-NS proteins by a phage protein.

H-NS family proteins, bacterial xenogeneic silencers, play central roles in genome organization and in the regulation of foreign genes. It is thought that gene repression is directly dependent on the DNA binding modes of H-NS family proteins. These proteins form lateral protofilaments along DNA. Under specific environmental conditions they switch to bridging two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the protein gp4, which modulates the DNA binding and function of the H-NS family protein MvaT of Pseudomonas aeruginosa. However, the mechanism by which gp4 affects MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT-DNA complex. Structural investigations suggest that gp4 acts as an 'electrostatic zipper' between the oppositely charged domains of MvaT protomers, and stabilizes a structure resembling their 'half-open' conformation, resulting in relief of gene silencing and adverse effects on P. aeruginosa growth. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.

Azetidomonamide and Diazetidomonapyridone Metabolites Control Biofilm Formation and Pigment Synthesis in Pseudomonas aeruginosa.

Synthesis of azetidine-derived natural products by the opportunistic pathogen Pseudomonas aeruginosa is controlled by quorum sensing, a process involving the production and sensing of diffusible signal molecules that is decisive for virulence regulation. In this study, we engineered P. aeruginosa for the titratable expression of the biosynthetic aze gene cluster, which allowed the purification and identification of two new products, azetidomonamide C and diazetidomonapyridone. Diazetidomonapyridone was shown to have a highly unusual structure with two azetidine rings and an open-chain diimide moiety. Expression of aze genes strongly increased biofilm formation and production of phenazine and alkyl quinolone virulence factors. Further physiological studies revealed that all effects were mainly mediated by azetidomonamide A and diazetidomonapyridone, whereas azetidomonamides B and C had little or no phenotypic impact. The P450 monooxygenase AzeF which catalyzes a challenging, stereoselective hydroxylation of the azetidine ring converting azetidomonamide C into azetidomonamide A is therefore crucial for biological activity. Based on our findings, we propose this group of metabolites to constitute a new class of diffusible regulatory molecules with community-related effects in P. aeruginosa.

Structural basis for osmotic regulation of the DNA binding properties of H-NS proteins.

H-NS proteins act as osmotic sensors translating changes in osmolarity into altered DNA binding properties, thus, regulating enterobacterial genome organization and genes transcription. The molecular mechanism underlying the switching process and its conservation among H-NS family members remains elusive. Here, we focus on the H-NS family protein MvaT from Pseudomonas aeruginosa and demonstrate experimentally that its protomer exists in two different conformations, corresponding to two different functional states. In the half-opened state (dominant at low salt) the protein forms filaments along DNA, in the fully opened state (dominant at high salt) the protein bridges DNA. This switching is a direct effect of ionic strength on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of MvaT. The asymmetric charge distribution and intramolecular interactions are conserved among the H-NS family of proteins. Therefore, our study establishes a general paradigm for the molecular mechanistic basis of the osmosensitivity of H-NS proteins.

Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade.

Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122-Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122-Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation.

Dynamics of Ligand Binding to a Rigid Glycosidase*.

The single-domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, which is one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, which is attributed to an enzyme-induced substrate distortion. A model is proposed in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates the acyl formation step of the hydrolysis reaction.

Uno Ferro, a de novo Designed Protein, Binds Transition Metals with High Affinity and Stabilizes Semiquinone Radical Anion.

Metalloenzymes often utilize radicals in order to facilitate chemical reactions. Recently, DeGrado and co-workers have discovered that model proteins can efficiently stabilize semiquinone radical anion produced by oxidation of 3,5-di-tert-butylcatechol (DTBC) in the presence of two zinc ions. Here, we show that the number and the nature of metal ions have relatively minor effect on semiquinone stabilization in model proteins, with a single metal ion being sufficient for radical stabilization. The radical is stabilized by both metal ion, hydrophobic sequestration, and interactions with the hydrophilic residues in the protein interior resulting in a remarkable, nearly 500 mV change in the redox potential of the SQ. - /catechol couple compared to bulk aqueous solution. Moreover, we have created 4G-UFsc, a single metal ion-binding protein with pm affinity for zinc that is higher than any other reported model systems and is on par with many natural zinc-containing proteins. We expect that the robust and easy-to-modify DFsc/UFsc family of proteins will become a versatile tool for mechanistic model studies of metalloenzymes.

In vivo biotinylated calpastatin improves the affinity purification of human m-calpain.

Recently we established a novel affinity purification method for calpain by exploiting the specific and reversible binding properties of its intrinsically disordered protein inhibitor, calpastatin. The immobilization strategy relied on the strength and specificity of the biotin - streptavidin interaction. Here, we report an improved and optimized method that even enables the general applicability of in vivo biotinylated (intrinsically disordered) proteins in any affinity capture strategy. Since in vitro chemical biotinylation is only accomplished with reagents that lack exact site specificity, it can not only cause sample heterogeneity but it can also hamper the functionality of the biotinylated molecules. Therefore, we have developed a recombinant expression protocol to produce in vivo biotinylated human calpastatin domain 1 (hCSD1) in Escherichia coli. We have experimentally verified that the biotinylated polypeptide tag is compatible with the intrinsically disordered state of hCSD1 and that it does not influence the functional properties of this intrinsically disordered protein (IDP). The in vivo biotinylated hCSD1 was then used without the need of any prepurification step prior to the affinity capturing of its substrate, human m-calpain. This leads to a simplified purification strategy that allows capturing the calpain efficiently from a complex biological mixture with only a single chromatogaphic step and in a considerably reduced timeframe. Our approach is generally applicable through the in vivo biotinylation of any IDP of interest, and its practical implementation will showcase the power to exploit the properties of IDPs in affinity capture strategies.

Anomer-Specific Recognition and Dynamics in a Fucose-Binding Lectin.

Sugar binding by a cell surface ∼29 kDa lectin (RSL) from the bacterium Ralstonia solanacearum was characterized by NMR spectroscopy. The complexes formed with four monosaccharides and four fucosides were studied. Complete resonance assignments and backbone dynamics were determined for RSL in the sugar-free form and when bound to l-fucose or d-mannose. RSL was found to interact with both the α- and the ß-anomer of l-fucose and the "fucose like" sugars d-arabinose and l-galactose. Peak splitting was observed for some resonances of the binding site residues. The assignment of the split signals to the α- or ß-anomer was confirmed by comparison with the spectra of RSL bound to methyl-α-l-fucoside or methyl-ß-l-fucoside. The backbone dynamics of RSL were sensitive to the presence of ligand, with the protein adopting a more compact structure upon binding to l-fucose. Taking advantage of tryptophan residues in the binding sites, we show that the indole resonance is an excellent reporter on ligand binding. Each sugar resulted in a distinct signature of chemical shift perturbations, suggesting that tryptophan signals are a sufficient probe of sugar binding.

Structure and Function of Transient Encounters of Redox Proteins.

Many biomolecular interactions proceed via lowly populated, transient intermediates. Believed to facilitate formation of a productive complex, these short-lived species are inaccessible to conventional biophysical and structural techniques and, until recently, could only be studied by theoretical simulations. Recent development of experimental approaches sensitive to the presence of minor species--in particular paramagnetic relaxation enhancement (PRE) NMR spectroscopy--has enabled direct visualization and detailed characterization of such lowly populated states. Collectively referred to as an encounter complex, the binding intermediates are particularly important in transient protein interactions, such as those orchestrating signaling cascades or energy-generating electron transfer (ET) chains. Here I discuss encounter complexes of redox proteins mediating biological ET reactions, which are essential for many vital cellular activities including oxidative phosphorylation and photosynthesis. In particular, this Account focuses on the complex of cytochrome c (Cc) and cytochrome c peroxidase (CcP), which is a paradigm of biomolecular ET and an attractive system for studying protein binding and enzymatic catalysis. The Cc-CcP complex formation proceeds via an encounter state, consisting of multiple protein-protein orientations sampled in the search of the dominant, functionally active bound form and exhibiting a broad spatial distribution, in striking agreement with earlier theoretical simulations. At low ionic strength, CcP binds another Cc molecule to form a weak ternary complex, initially inferred from kinetics experiments and postulated to account for the measured ET activity. Despite strenuous efforts, the ternary complex could not be observed directly and remained eagerly sought for the past two decades. Very recently, we have solved its structure in solution and shown that it consists of two binding forms: the dominant, ET-inactive geometry and an ensemble of lowly populated species with short separations between Cc and CcP cofactors, which summarily account for the measured ET rate. Unlike most protein complexes, which require accurate alignment of the binding surfaces in a single, well-defined orientation to carry out their function, redox proteins can form multiple productive complexes. As fast ET will occur any time the redox centers of the binding partners are close enough to ensure efficient electron tunneling across the interface, many protein-protein orientations are expected to be ET active. The present analysis confirms that the low-occupancy states can support the functional ET activity and contribute to the stability of redox protein complexes. As illustrated here, boundaries between the dominant and the encounter forms become blurred for many dynamic ET systems, which are more aptly described by ensembles of functionally and structurally heterogeneous bound forms.

Cofactor-dependent structural and binding properties of yeast cytochrome C peroxidase.

Yeast cytochrome c peroxidase (CcP) is a heme enzyme that reduces hydroperoxides using the electrons provided by its physiological partner cytochrome c (Cc). Contributing to the resistance against the oxidative stress associated with the aerobic metabolism, the Cc-CcP complex has been widely studied and became a paradigm for biological electron transfer. The heme-free, enzymatically inactive apo CcP is the natural precursor of the mature, cofactor-bound holo protein. Despite its physiological relevance, apo CcP is not well characterized, and at present, little is known about its structure or the interaction with Cc. Using a range of biophysical techniques, here we show that, while holo CcP binds Cc with micromolar affinity, the interaction between apo CcP and Cc is completely abolished. Characterized by small-angle X-ray scattering, solution nuclear magnetic resonance spectroscopy, and equilibrium unfolding experiments, apo and holo CcP exhibit very similar structural, hydrodynamic, and thermodynamic properties. However, detailed analysis reveals that apo CcP is more expanded in solution, displays a number of characteristics associated with a molten globule state, and, unlike the holo protein, does not form an unfolding intermediate during thermal and chemical denaturation. Overall, our data suggest that the Cc binding site present in the holo protein is disrupted in the apo form, explaining the inability of the latter to interact with Cc. We argue that the observed difference in Cc binding is physiologically relevant and suggest why abolishing the apo CcP-Cc interaction is beneficial to the organism.

Toxin:antitoxin ratio sensing autoregulation of the Vibrio cholerae parDE2 module.

The parDE family of toxin-antitoxin (TA) operons is ubiquitous in bacterial genomes and, in Vibrio cholerae, is an essential component to maintain the presence of chromosome II. Here, we show that transcription of the V. cholerae parDE2 (VcparDE) operon is regulated in a toxin:antitoxin ratio-dependent manner using a molecular mechanism distinct from other type II TA systems. The repressor of the operon is identified as an assembly with a 6:2 stoichiometry with three interacting ParD2 dimers bridged by two ParE2 monomers. This assembly docks to a three-site operator containing 5'- GGTA-3' motifs. Saturation of this TA complex with ParE2 toxin results in disruption of the interface between ParD2 dimers and the formation of a TA complex of 2:2 stoichiometry. The latter is operator binding-incompetent as it is incompatible with the required spacing of the ParD2 dimers on the operator.

Fuzzy recognition by the prokaryotic transcription factor HigA2 from Vibrio cholerae.

Disordered protein sequences can exhibit different binding modes, ranging from well-ordered folding-upon-binding to highly dynamic fuzzy binding. The primary function of the intrinsically disordered region of the antitoxin HigA2 from Vibrio cholerae is to neutralize HigB2 toxin through ultra-high-affinity folding-upon-binding interaction. Here, we show that the same intrinsically disordered region can also mediate fuzzy interactions with its operator DNA and, through interplay with the folded helix-turn-helix domain, regulates transcription from the higBA2 operon. NMR, SAXS, ITC and in vivo experiments converge towards a consistent picture where a specific set of residues in the intrinsically disordered region mediate electrostatic and hydrophobic interactions while "hovering" over the DNA operator. Sensitivity of the intrinsically disordered region to scrambling the sequence, position-specific contacts and absence of redundant, multivalent interactions, point towards a more specific type of fuzzy binding. Our work demonstrates how a bacterial regulator achieves dual functionality by utilizing two distinct interaction modes within the same disordered sequence.

Biophysical characterization of the Plasmodium falciparum circumsporozoite protein's N-terminal domain.

The circumsporozoite protein (CSP) is the main surface antigen of the Plasmodium sporozoite (SPZ) and forms the basis of the currently only licensed anti-malarial vaccine (RTS,S/AS01). CSP uniformly coats the SPZ and plays a pivotal role in its immunobiology, in both the insect and the vertebrate hosts. Although CSP's N-terminal domain (CSPN ) has been reported to play an important role in multiple CSP functions, a thorough biophysical and structural characterization of CSPN is currently lacking. Here, we present an alternative method for the recombinant production and purification of CSPN from Plasmodium falciparum (PfCSPN ), which provides pure, high-quality protein preparations with high yields. Through an interdisciplinary approach combining in-solution experimental methods and in silico analyses, we provide strong evidence that PfCSPN is an intrinsically disordered region displaying some degree of compaction.

Expression, purification, characterization, and solution nuclear magnetic resonance study of highly deuterated yeast cytochrome C peroxidase with enhanced solubility.

Here we present the preparation, biophysical characterization, and nuclear magnetic resonance (NMR) spectroscopy study of yeast cytochrome c peroxidase (CcP) constructs with enhanced solubility. Using a high-yield Escherichia coli expression system, we routinely produced uniformly labeled [(2)H,(13)C,(15)N]CcP samples with high levels of deuterium incorporation (96-99%) and good yields (30-60 mg of pure protein from 1 L of bacterial culture). In addition to simplifying the purification procedure, introduction of a His tag at either protein terminus dramatically increases its solubility, allowing preparation of concentrated, stable CcP samples required for multidimensional NMR spectroscopy. Using a range of biophysical techniques and X-ray crystallography, we demonstrate that the engineered His tags neither perturb the structure of the enzyme nor alter the heme environment or its reactivity toward known ligands. The His-tagged CcP constructs remain catalytically active yet exhibit differences in the interaction with cytochrome c, the physiological binding partner, most likely because of steric occlusion of the high-affinity binding site by the C-terminal His tag. We show that protein perdeuteration greatly increases the quality of the double- and triple-resonance NMR spectra, allowing nearly complete backbone resonance assignments and subsequent study of the CcP by heteronuclear NMR spectroscopy.