Structure-based Mutagenesis Studies Research Articles

Substrate recognition by the 4-hydroxytryptamine kinase PsiK in psilocybin biosynthesis.

Psilocybin, the natural hallucinogen from Psilocybe (magic) mushrooms, is a highly promising drug candidate for the treatment of depression and several other mental health conditions. Biosynthesis of psilocybin from the amino acid l-tryptophan involves four strictly sequential modifications. The third of these, ATP-dependent phosphorylation of the intermediate 4-hydroxytryptamine, is catalysed by PsiK. Here we present a crystallographic analysis and a structure-based mutagenesis study of this kinase, providing insight into its mode of substrate recognition. The results of our work will support future bioengineering efforts aimed at generating variants of psilocybin with enhanced therapeutic properties.

The SUN-family protein Sad1 mediates heterochromatin spatial organization through interaction with histone H2A-H2B

Heterochromatin is generally associated with the nuclear periphery, but how the spatial organization of heterochromatin is regulated to ensure epigenetic silencing remains unclear. Here we found that Sad1, an inner nuclear membrane SUN-family protein in fission yeast, interacts with histone H2A-H2B but not H3-H4. We solved the crystal structure of the histone binding motif (HBM) of Sad1 in complex with H2A-H2B, revealing the intimate contacts between Sad1HBM and H2A-H2B. Structure-based mutagenesis studies revealed that the H2A-H2B-binding activity of Sad1 is required for the dynamic distribution of Sad1 throughout the nuclear envelope (NE). The Sad1-H2A-H2B complex mediates tethering telomeres and the mating-type locus to the NE. This complex is also important for heterochromatin silencing. Mechanistically, H2A-H2B enhances the interaction between Sad1 and HDACs, including Clr3 and Sir2, to maintain epigenetic identity of heterochromatin. Interestingly, our results suggest that Sad1 exhibits the histone-enhanced liquid-liquid phase separation property, which helps recruit heterochromatin factors to the NE. Our results uncover an unexpected role of SUN-family proteins in heterochromatin regulation and suggest a nucleosome-independent role of H2A-H2B in regulating Sad1’s functionality.

Structural basis for botulinum neurotoxin E recognition of synaptic vesicle protein 2

Botulinum neurotoxin E (BoNT/E) is one of the major causes of human botulism and paradoxically also a promising therapeutic agent. Here we determined the co-crystal structures of the receptor-binding domain of BoNT/E (HCE) in complex with its neuronal receptor synaptic vesicle glycoprotein 2A (SV2A) and a nanobody that serves as a ganglioside surrogate. These structures reveal that the protein-protein interactions between HCE and SV2 provide the crucial location and specificity information for HCE to recognize SV2A and SV2B, but not the closely related SV2C. At the same time, HCE exploits a separated sialic acid-binding pocket to mediate recognition of an N-glycan of SV2. Structure-based mutagenesis and functional studies demonstrate that both the protein-protein and protein-glycan associations are essential for SV2A-mediated cell entry of BoNT/E and for its potent neurotoxicity. Our studies establish the structural basis to understand the receptor-specificity of BoNT/E and to engineer BoNT/E variants for new clinical applications.

Structural basis for the transformation of the traditional medicine berberine by bacterial nitroreductase.

The bacterial nitroreductases (NRs) NfsB and NfsA are conserved homodimeric FMN-dependent flavoproteins that are responsible for the reduction of nitroaromatic substrates. Berberine (BBR) is a plant-derived isoquinoline alkaloid with a large conjugated ring system that is widely used in the treatment of various diseases. It was recently found that the gut microbiota convert BBR into dihydroberberine (dhBBR, the absorbable form) mediated by bacterial NRs. The molecular basis for the transformation of BBR by the gut microbiota remains unclear. Here, kinetic studies showed that NfsB from Escherichia coli (EcNfsB), rather than EcNfsA, is responsible for the conversion of BBR to dhBBR in spite of a low reaction rate. The crystal structure of the EcNfsB-BBR complex showed that BBR binds into the active pocket at the dimer interface, and its large conjugated plane stacks above the plane of the FMN cofactor in a nearly parallel orientation. BBR is mainly stabilized by π-stacking interactions with both neighboring aromatic residues and FMN. Structure-based mutagenesis studies further revealed that the highly conserved Phe70 and Phe199 are important residues for the conversion of BBR. The structure revealed that the C6 atom of BBR (which receives the hydride) is ∼7.5 Å from the N5 atom of FMN (which donates the hydride), which is too distant for hydride transfer. Notably, several well ordered water molecules make hydrogen-bond/van der Waals contacts with the N1 atom of BBR in the active site, which probably donate protons in conjunction with electron transfer from FMN. The structure-function studies revealed the mechanism for the recognition and binding of BBR by bacterial NRs and may help to understand the conversion of BBR by the gut microbiota.

Molecular insights into the unusually promiscuous and catalytically versatile Fe(II)/\u03b1-ketoglutarate-dependent oxygenase SptF

Non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases catalyze various oxidative biotransformations. Due to their catalytic flexibility and high efficiency, Fe/αKG oxygenases have attracted keen attention for their application as biocatalysts. Here, we report the biochemical and structural characterizations of the unusually promiscuous and catalytically versatile Fe/αKG oxygenase SptF, involved in the biosynthesis of fungal meroterpenoid emervaridones. The in vitro analysis revealed that SptF catalyzes several continuous oxidation reactions, including hydroxylation, desaturation, epoxidation, and skeletal rearrangement. SptF exhibits extremely broad substrate specificity toward various meroterpenoids, and efficiently produced unique cyclopropane-ring-fused 5/3/5/5/6/6 and 5/3/6/6/6 scaffolds from terretonins. Moreover, SptF also hydroxylates steroids, including androsterone, testosterone, and progesterone, with different regiospecificities. Crystallographic and structure-based mutagenesis studies of SptF revealed the molecular basis of the enzyme reactions, and suggested that the malleability of the loop region contributes to the remarkable substrate promiscuity. SptF exhibits great potential as a promising biocatalyst for oxidation reactions.

Mammalian ALKBH1 serves as an N6-mA demethylase of unpairing DNA

N6-methyladenine (N6-mA) of DNA is an emerging epigenetic mark in mammalian genome. Levels of N6-mA undergo drastic fluctuation during early embryogenesis, indicative of active regulation. Here we show that the 2-oxoglutarate-dependent oxygenase ALKBH1 functions as a nuclear eraser of N6-mA in unpairing regions (e.g., SIDD, Stress-Induced DNA Double Helix Destabilization regions) of mammalian genomes. Enzymatic profiling studies revealed that ALKBH1 prefers bubbled or bulged DNAs as substrate, instead of single-stranded (ss-) or double-stranded (ds-) DNAs. Structural studies of ALKBH1 revealed an unexpected “stretch-out” conformation of its “Flip1” motif, a conserved element that usually bends over catalytic center to facilitate substrate base flipping in other DNA demethylases. Thus, lack of a bending “Flip1” explains the observed preference of ALKBH1 for unpairing substrates, in which the flipped N6-mA is primed for catalysis. Co-crystal structural studies of ALKBH1 bound to a 21-mer bulged DNA explained the need of both flanking duplexes and a flipped base for recognition and catalysis. Key elements (e.g., an ALKBH1-specific α1 helix) as well as residues contributing to structural integrity and catalytic activity were validated by structure-based mutagenesis studies. Furthermore, ssDNA-seq and DIP-seq analyses revealed significant co-occurrence of base unpairing regions with N6-mA in mouse genome. Collectively, our biochemical, structural and genomic studies suggest that ALKBH1 is an important DNA demethylase that regulates genome N6-mA turnover of unpairing regions associated with dynamic chromosome regulation.

Structural insights into the catalytic mechanism of lovastatin hydrolase

Structural insights into the catalytic mechanism of lovastatin hydrolase

Structural basis of species-selective antagonist binding to the succinate receptor.

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3-5. Because SUCNR1 senses succinate as an immunological danger signal6-which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8-it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.

Cryo-EM Studies of TMEM16F Calcium-Activated Ion Channel Suggest Features Important for Lipid Scrambling

SUMMARYAs a Ca2+-activated lipid scramblase and ion channel that mediates Ca2+ influx, TMEM16F relies on both functions to facilitate extracellular vesicle generation, blood coagulation, and bone formation. How a bona fide ion channel scrambles lipids remains elusive. Our structural analyses revealed the coexistence of an intact channel pore and PIP2-dependent protein conformation changes leading to membrane distortion. Correlated to the extent of membrane distortion, many tightly bound lipids are slanted. Structure-based mutagenesis studies further reveal that neutralization of some lipid-binding residues or those near membrane distortion specifically alters the onset of lipid scrambling, but not Ca2+ influx, thus identifying features outside of channel pore that are important for lipid scrambling. Together, our studies demonstrate that membrane distortion does not require open hydrophilic grooves facing the membrane interior and provide further evidence to suggest separate pathways for lipid scrambling and ion permeation.

Identification of UDP-glucose binding site in glycosyltransferase domain of sucrose phosphate synthase from sugarcane (Saccharum officinarum) by structure-based site-directed mutagenesis.

Sucrose phosphate synthase (SPS) is believed to be the key enzyme for controlling the biosynthesis of sucrose. SPSs consist of a functional glycosyltransferase domain that shares conserved residues with the glycosyltransferase domain of sucrose biosynthesis-related protein. The formation of sucrose-6-phosphate is catalyzed by SPS with the transfer of a glycosyl group of uridine diphosphate glucose (UDP-G) as an activated donor sugar to a fructose-6-phosphate as a sugar acceptor. However, understanding of the mechanism of catalytic and substrate binding in SPS is very limited. Based on amino acid sequence alignments with several enzymes that belong to the glycosyltransferase family, the UDP-G binding sites that might be critical for catalytic mechanism were identified. Here, we report that single point mutation of R496, D498, and V570 located in the proposed UDP-G binding site led to less active or complete loss of enzyme activity. Through structure-based site-directed mutagenesis and biochemical studies, the results indicated that these residues contribute to the catalytic activity of plant SPS. Moreover, understanding of the UDP-G binding site provides an insight into new strategies for enzyme engineering and redesigning a catalytic mechanism for UDP.

A structural comparison of Listeria monocytogenes protein chaperones PrsA1 and PrsA2 reveals molecular features required for virulence.

Listeria monocytogenes is a Gram-positive environmental bacterium that lives within soil but transitions into a pathogen upon contact with a mammalian host. The transition of L. monocytogenes from soil dweller to cytosolic pathogen is dependent upon secreted virulence factors that mediate cell invasion and intracellular growth. PrsA1 and PrsA2 are secreted bacterial lipoprotein chaperones that contribute to the folding of proteins translocated across the bacterial membrane; PrsA2 is required for L. monocytogenes virulence, whereas the function of PrsA1 remains to be determined. We have solved an X-ray crystal structure of PrsA1 and have used this model to guide comparison structure-based mutagenesis studies with PrsA2. Targeted mutagenesis of PrsA2 demonstrates that oligomerization of PrsA2 as well as molecular features of the foldase domain are required for protein secretion and virulence, whereas a functional role was uncovered for PrsA1 in bacterial resistance to alcohol. Interestingly, PrsA2 membrane localization is not required for all PrsA2-dependent activities, suggesting that the lipoprotein retains function when released from the bacterial cell. PrsA chaperones are thus multifaceted proteins with distinct domains adapted to accommodate the functional needs of a diverse array of secreted substrates.

Atomic Structure of a Non-Enveloped dsRNA Virus Reveals pH Sensing for Cell Entry

Mechanism to monitor and response to environmental changes by non-enveloped dsRNA viruses during cell entry is largely unclear, which is in contrast to that by envelope viruses. In particular, how these viruses sense pH change inside endosomes and disrupt host membrane to gain entry into cytoplasm are not understood. To gain insight into cell entry by non-enveloped dsRNA viruses, we determined the atomic structure of the bluetongue virus (BTV) at pH 8.5, a non-enveloped dsRNA virus that undergoes a two-stage endosomal process of cell entry. Our structure reveals that the receptor-binding protein VP2 possesses a conserved zinc-finger motif which may be sensitive to low pH, and that the membrane penetration protein VP5 has three domains: dagger, unfurling and anchoring. The core of the anchoring domain not only contains a β-meander motif with a histidine cluster for low-pH sensing, but also has features potentially for membrane interactions: aromatic clusters, a hydrophobic surface and a WHXL motif. The cryoEM structure of BTV at low pH (5.5 & 3.4) reveals that the VP2 detaches from virus particles, and that the VP5 undergoes dramatic conformational change with the dagger and unfurling domains of VP5 trimer project outwards to form a long filament structure while the anchoring domain remains attached on virus inner capsid. Consistently, biochemical and structure-based mutagenesis studies support these mechanistic interpretations.

Structure-Based Mechanism of Oleate Hydratase from Elizabethkingia meningoseptica.

Hydratases provide access to secondary and tertiary alcohols by regio- and/or stereospecifically adding water to carbon-carbon double bonds. Thereby, hydroxy groups are introduced without the need for costly cofactor recycling, and that makes this approach highly interesting on an industrial scale. Here we present the first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD). A structure-based mutagenesis study targeting active site residues identified E122 and Y241 as crucial for the activation of a water molecule and for protonation of the double bond, respectively. Moreover, we also observed that two-electron reduction of FAD results in a sevenfold increase in the substrate hydration rate. We propose the first reaction mechanism for this enzyme class that explains the requirement for the flavin cofactor and the involvement of conserved amino acid residues in this regio- and stereoselective hydration.

Crystal structure of Bax bound to the BH3 peptide of Bim identifies important contacts for interaction.

The BH3-only protein Bim is a potent direct activator of the proapoptotic effector protein Bax, but the structural basis for its activity has remained poorly defined. Here we describe the crystal structure of the BimBH3 peptide bound to BaxΔC26 and structure-based mutagenesis studies. Similar to BidBH3, the BimBH3 peptide binds into the cognate surface groove of Bax using the conserved hydrophobic BH3 residues h1–h4. However, the structure and mutagenesis data show that Bim is less reliant compared with Bid on its ‘h0' residues for activating Bax and that a single amino-acid difference between Bim and Bid encodes a fivefold difference in Bax-binding potency. Similar to the structures of BidBH3 and BaxBH3 bound to BaxΔC21, the structure of the BimBH3 complex with BaxΔC displays a cavity surrounded by Bax α1, α2, α5 and α8. Our results are consistent with a model in which binding of an activator BH3 domain to the Bax groove initiates separation of its core (α2–α5) and latch (α6–α8) domains, enabling its subsequent dimerisation and the permeabilisation of the mitochondrial outer membrane.

Functional and Structural Characterization of the Antiphagocytic Properties of a Novel Transglutaminase from Streptococcus suis

Streptococcus suis serotype 2 (Ss2) is an important swine and human zoonotic pathogen. In the present study, we identified a novel secreted immunogenic protein, SsTGase, containing a highly conserved eukaryotic-like transglutaminase (TGase) domain at the N terminus. We found that inactivation of SsTGase significantly reduced the virulence of Ss2 in a pig infection model and impaired its antiphagocytosis in human blood. We further solved the crystal structure of the N-terminal portion of the protein in homodimer form at 2.1 Å. Structure-based mutagenesis and biochemical studies suggested that disruption of the homodimer directly resulted in the loss of its TGase activity and antiphagocytic ability. Characterization of SsTGase as a novel virulence factor of Ss2 by acting as a TGase would be beneficial for developing new therapeutic agents against Ss2 infections.

A Photobacterium sp. α2–6-sialyltransferase (Psp2,6ST) mutant with an increased expression level and improved activities in sialylating Tn antigens

In order to improve the catalytic efficiency of recombinant Photobacterium sp. JT-ISH-224 α2–6-sialyltransferase Psp2,6ST(15–501)-His6 in sialylating α-GalNAc-containing acceptors for the synthesis of tumor-associated carbohydrate antigens sialyl Tn (STn), protein crystal structure-based mutagenesis studies were carried out. Among several mutants obtained by altering the residues close to the acceptor substrate binding pocket, mutant A366G was shown to improve the sialyltransferase activity of Psp2,6ST(15–501)-His6 toward α-GalNAc-containing acceptors by 21–115% without significantly affecting its sialylation activity to β-galactosides. Furthermore, the expression level was improved from 18–40mgL−1 for the wild-type enzyme to 72–110mgL−1 for the A366G mutant. In situ generation of CMP-sialic acid in a one-pot two-enzyme system was shown effective in overcoming the high donor hydrolysis of the enzyme. Mutant A366G performed better than the wild-type Psp2,6ST(15–501)-His6 for synthesizing Neu5Acα2–6GalNAcαOSer/Thr STn antigens.

Structure-based functional characterization of repressor of toxin (Rot), a central regulator of Staphylococcus aureus virulence.

Staphylococcus aureus is responsible for a large number of diverse infections worldwide. In order to support its pathogenic lifestyle, S. aureus has to regulate the expression of virulence factors in a coordinated fashion. One of the central regulators of the S. aureus virulence regulatory networks is the transcription factor repressor of toxin (Rot). Rot plays a key role in regulating S. aureus virulence through activation or repression of promoters that control expression of a large number of critical virulence factors. However, the mechanism by which Rot mediates gene regulation has remained elusive. Here, we have determined the crystal structure of Rot and used this information to probe the contribution made by specific residues to Rot function. Rot was found to form a dimer, with each monomer harboring a winged helix-turn-helix (WHTH) DNA-binding motif. Despite an overall acidic pI, the asymmetric electrostatic charge profile suggests that Rot can orient the WHTH domain to bind DNA. Structure-based site-directed mutagenesis studies demonstrated that R(91), at the tip of the wing, plays an important role in DNA binding, likely through interaction with the minor groove. We also found that Y(66), predicted to bind within the major groove, contributes to Rot interaction with target promoters. Evaluation of Rot binding to different activated and repressed promoters revealed that certain mutations on Rot exhibit promoter-specific effects, suggesting for the first time that Rot differentially interacts with target promoters. This work provides insight into a precise mechanism by which Rot controls virulence factor regulation in S. aureus.

Structural and functional analysis of the β‐barrel domain of BamA from Escherichia coli

In gram-negative bacteria, the assembly of outer membrane proteins (OMPs) requires a β-barrel assembly machinery (BAM) complex, of which BamA is an essential and evolutionarily conserved component. To elucidate the mechanism of BamA-mediated OMP biogenesis, we determined the crystal structure of the C-terminal transmembrane domain of BamA from Escherichia coli (EcBamA) at 2.6 Å resolution. The structure reveals 2 distinct features. First, a portion of the extracellular side of the β barrel is composed of 5 markedly short β strands, and the loops stemming from these β strands form a potential surface cavity, filled by a portion of the L6 loop that includes the conserved VRGF/Y motif found in the Omp85 family. Second, the 4 extracellular loops L3, L4, L6, and L7 of EcBamA form a dome over the barrel, stabilized by a salt-bridge interaction network. Functional data show that hydrophilic-to-hydrophobic mutations of the potential hydrophilic surface cavity and a single Arg547Ala point mutation that may destabilize the dome severely affect the function of EcBamA. Our structure of the EcBamA β barrel and structure-based mutagenesis studies suggest that the transmembrane β strands of OMP substrates may integrate into the outer membrane at the interface of the first and last β strands of the EcBamA barrel, whereas the soluble loops or domains may be transported out of the cell via the hydrophilic surface cavity on dislocation of the VRGF/Y motif of L6. In addition, the dome over the barrel may play an important role in maintaining the efficiency of OMP biogenesis.

Structure of the HopA1(21-102)-ShcA Chaperone-Effector Complex of Pseudomonas syringae Reveals Conservation of a Virulence Factor Binding Motif from Animal to Plant Pathogens

Pseudomonas syringae injects numerous bacterial proteins into host plant cells through a type 3 secretion system (T3SS). One of the first such bacterial effectors discovered, HopA1, is a protein that has unknown functions in the host cell but possesses close homologs that trigger the plant hypersensitive response in resistant strains. Like the virulence factors in many bacterial pathogens of animals, HopA1 depends upon a cognate chaperone in order to be effectively translocated by the P. syringae T3SS. Herein, we report the crystal structure of a complex of HopA1(21-102) with its chaperone, ShcA, determined to 1.56-Å resolution. The structure reveals that three key features of the chaperone-effector interactions found in animal pathogens are preserved in the Gram-negative pathogens of plants, namely, (i) the interaction of the chaperone with a nonglobular polypeptide of the effector, (ii) an interaction centered on the so-called β-motif, and (iii) the presence of a conserved hydrophobic patch in the chaperone that recognizes the β-motif. Structure-based mutagenesis and biochemical studies have established that the β-motif is critical for the stability of this complex. Overall, these results show that the β-motif interactions are broadly conserved in bacterial pathogens utilizing T3SSs, spanning an interkingdom host range.

Structure of the α-tubulin acetyltransferase, αTAT1, and implications for tubulin-specific acetylation

Protein acetylation is an important posttranslational modification with the recent identification of new substrates and enzymes, new links to disease, and modulators of protein acetylation for therapy. α-Tubulin acetyltransferase (αTAT1) is the major α-tubulin lysine-40 (K40) acetyltransferase in mammals, nematodes, and protozoa, and its activity plays a conserved role in several microtubule-based processes. Here, we present the X-ray crystal structure of the human αTAT1/acetyl-CoA complex. Together with structure-based mutagenesis, enzymatic analysis, and functional studies in cells, we elucidate the catalytic mechanism and mode of tubulin-specific acetylation. We find that αTAT1 has an overall fold similar to the Gcn5 histone acetyltransferase but contains a relatively wide substrate binding groove and unique structural elements that play important roles in α-tubulin-specific acetylation. Conserved aspartic acid and cysteine residues play important catalytic roles through a ternary complex mechanism. αTAT1 mutations have analogous effects on tubulin acetylation in vitro and in cells, demonstrating that it is the central determining factor of α-tubulin K40 acetylation levels in vivo. Together, these studies provide general insights into distinguishing features between histone and tubulin acetyltransferases, and they have specific implications for understanding the molecular basis of tubulin acetylation and for developing small molecule modulators of microtubule acetylation for therapy.