Structural Characterization of Disulfide-Linked p53-Derived Peptide Dimers.

Disulfide bonds provide a convenient method for chemoselective alteration of peptide and protein structure and function. We previously reported that mild oxidation of a p53-derived bisthiol peptide (CTFANLWRLLAQNC) under dilute non-denaturing conditions led to unexpected disulfide-linked dimers as the exclusive product. The dimers were antiparallel, significantly α-helical, resistant to protease degradation, and easily reduced back to the original bisthiol peptide. Here we examine the intrinsic factors influencing peptide dimerization using a combination of amino acid substitution, circular dichroism (CD) spectroscopy, and X-ray crystallography. CD analysis of peptide variants suggests critical roles for Leu6 and Leu10 in the formation of stable disulfide-linked dimers. The 1.0 Å resolution crystal structure of the peptide dimer supports these data, revealing a leucine-rich LxxLL dimer interface with canonical knobs-into-holes packing. Two levels of higher-order oligomerization are also observed in the crystal: an antiparallel "dimer of dimers" mediated by Phe3 and Trp7 residues in the asymmetric unit and a tetramer of dimers mediated by Trp7 and Leu10. In CD spectra of Trp-containing peptide variants, minima at 227 nm provide evidence for the dimer of dimers in dilute aqueous solution. Importantly, and in contrast to the original dimer model, the canonical leucine-rich core and robust dimerization of most peptide variants suggests a tunable molecular architecture to target various proteins and evaluate how folding and oligomerization impact various properties, such as cell permeability.

Bragg Spot Finder (BSF): a new machine-learning-aided approach to deal with spot finding for rapidly filtering diffraction pattern images.

Macromolecular crystallography contributes significantly to understanding diseases and, more importantly, how to treat them by providing atomic resolution 3D structures of proteins. This is achieved by collecting X-ray diffraction images of protein crystals from important biological pathways. Spotfinders are used to detect the presence of crystals with usable data, and the spots from such crystals are the primary data used to solve the relevant structures. Having fast and accurate spot finding is essential, but recent advances in synchrotron beamlines used to generate X-ray diffraction images have brought us to the limits of what the best existing spotfinders can do. This bottleneck must be removed so spotfinder software can keep pace with the X-ray beamline hardware improvements and be able to see the weak or diffuse spots required to solve the most challenging problems encountered when working with diffraction images. In this paper, we first present Bragg Spot Detection (BSD), a large benchmark Bragg spot image dataset that contains 304 images with more than 66 000 spots. We then discuss the open source extensible U-Net-based spotfinder Bragg Spot Finder (BSF), with image pre-processing, a U-Net segmentation backbone, and post-processing that includes artifact removal and watershed segmentation. Finally, we perform experiments on the BSD benchmark and obtain results that are (in terms of accuracy) comparable to or better than those obtained with two popular spotfinder software packages (Dozor and DIALS), demonstrating that this is an appropriate framework to support future extensions and improvements.

A hemoprotein with a zinc-mirror heme site ties heme availability to carbon metabolism in cyanobacteria.

Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site. Together with structure-guided in vitro and in vivo experiments, we further demonstrate the existence of a functional link between heme binding by Dri1 (Domain related to iron 1, formerly ssr1698) and post-translational regulation of succinate dehydrogenase in the cyanobacterium Synechocystis, suggesting an iron-dependent regulatory link between photosynthesis and respiration. Given the ubiquity of proteins containing homologous domains and connections to heme metabolism across eukaryotes and prokaryotes, we propose that DRI (Domain Related to Iron; formerly DUF2470) functions at the molecular level as a heme-dependent regulatory domain.

Activation of CBASS Cap5 endonuclease immune effector by cyclic nucleotides.

The bacterial cyclic oligonucleotide-based antiphage signaling system (CBASS) is similar to the cGAS-STING system in humans, containing an enzyme that synthesizes a cyclic nucleotide on viral infection and an effector that senses the second messenger for the antiviral response. Cap5, containing a SAVED domain coupled to an HNH DNA endonuclease domain, is the most abundant CBASS effector, yet the mechanism by which it becomes activated for cell killing remains unknown. We present here high-resolution structures of full-length Cap5 from Pseudomonas syringae (Ps) with second messengers. The key to PsCap5 activation is a dimer-to-tetramer transition, whereby the binding of second messenger to dimer triggers an open-to-closed transformation of the SAVED domains, furnishing a surface for assembly of the tetramer. This movement propagates to the HNH domains, juxtaposing and converting two HNH domains into states for DNA destruction. These results show how Cap5 effects bacterial cell suicide and we provide proof-in-principle data that the CBASS can be extrinsically activated to limit bacterial infections.

Harnessing Aromatic-Histidine Interactions through Synergistic Backbone Extension and Side Chain Modification.

Peptide engineering efforts have delivered drugs for diverse human diseases. Side chain alteration is among the most common approaches to designing new peptides for specific applications. The peptide backbone can be modified as well, but this strategy has received relatively little attention. Here we show that new and favorable contacts between a His side chain on a target protein and an aromatic side chain on a synthetic peptide ligand can be engineered by rational and coordinated side chain modification and backbone extension. Side chain modification alone was unsuccessful. Binding measurements, high-resolution structural studies and pharmacological outcomes all support the synergy between backbone and side chain modification in engineered ligands of the parathyroid hormone receptor-1, which is targeted by osteoporosis drugs. These results should motivate other structure-based designs featuring coordinated side chain modification and backbone extension to enhance the engagement of peptide ligands with target proteins.

EstG is a novel esterase required for cell envelope integrity in Caulobacter.

Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the ⍺-proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell-wall-targeting antibiotics, EstG does not demonstrate biochemical activity toward cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which are important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.

AMX - the highly automated macromolecular crystallography (17-ID-1) beamline at the NSLS-II.

The highly automated macromolecular crystallography beamline AMX/17-ID-1 is an undulator-based high-intensity (>5 × 1012 photons s-1), micro-focus (7 µm × 5 µm), low-divergence (1 mrad × 0.35 mrad) energy-tunable (5-18 keV) beamline at the NSLS-II, Brookhaven National Laboratory, Upton, NY, USA. It is one of the three life science beamlines constructed by the NIH under the ABBIX project and it shares sector 17-ID with the FMX beamline, the frontier micro-focus macromolecular crystallography beamline. AMX saw first light in March 2016 and started general user operation in February 2017. At AMX, emphasis has been placed on high throughput, high capacity, and automation to enable data collection from the most challenging projects using an intense micro-focus beam. Here, the current state and capabilities of the beamline are reported, and the different macromolecular crystallography experiments that are routinely performed at AMX/17-ID-1 as well as some plans for the near future are presented.

The ribosomal RNA processing 1B:protein phosphatase 1 holoenzyme reveals non-canonical PP1 interaction motifs.

The serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of substrates by associating with >200 regulatory proteins to form specific holoenzymes. The major PP1 targeting protein in the nucleolus is RRP1B (ribosomal RNA processing 1B). In addition to selectively recruiting PP1ß/PP1γ to the nucleolus, RRP1B also has a key role in ribosome biogenesis, among other functions. How RRP1B binds PP1 and regulates nucleolar phosphorylation signaling is not yet known. Here, we show that RRP1B recruits PP1 via established (RVxF/SILK/ΦΦ) and non-canonical motifs. These atypical interaction sites, the PP1ß/γ specificity, and N-terminal AF-binding pockets rely on hydrophobic interactions that contribute to binding and, via phosphorylation, regulate complex formation. This work advances our understanding of PP1 isoform selectivity, reveals key roles of N-terminal PP1 residues in regulator binding, and suggests that additional PP1 interaction sites have yet to be identified, all of which are necessary for a systems biology understanding of PP1 function.

Hepatitis C virus NS3/4A inhibitors and other drug-like compounds as covalent binders of SARS-CoV-2 main protease.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), threatens global public health. The world needs rapid development of new antivirals and vaccines to control the current pandemic and to control the spread of the variants. Among the proteins synthesized by the SARS-CoV-2 genome, main protease (Mpro also known as 3CLpro) is a primary drug target, due to its essential role in maturation of the viral polyproteins. In this study, we provide crystallographic evidence, along with some binding assay data, that three clinically approved anti hepatitis C virus drugs and two other drug-like compounds covalently bind to the Mpro Cys145 catalytic residue in the active site. Also, molecular docking studies can provide additional insight for the design of new antiviral inhibitors for SARS-CoV-2 using these drugs as lead compounds. One might consider derivatives of these lead compounds with higher affinity to the Mpro as potential COVID-19 therapeutics for further testing and possibly clinical trials.

A simple technique to classify diffraction data from dynamic proteins according to individual polymorphs.

One often observes small but measurable differences in the diffraction data measured from different crystals of a single protein. These differences might reflect structural differences in the protein and may reveal the natural dynamism of the molecule in solution. Partitioning these mixed-state data into single-state clusters is a critical step that could extract information about the dynamic behavior of proteins from hundreds or thousands of single-crystal data sets. Mixed-state data can be obtained deliberately (through intentional perturbation) or inadvertently (while attempting to measure highly redundant single-crystal data). To the extent that different states adopt different molecular structures, one expects to observe differences in the crystals; each of the polystates will create a polymorph of the crystals. After mixed-state diffraction data have been measured, deliberately or inadvertently, the challenge is to sort the data into clusters that may represent relevant biological polystates. Here, this problem is addressed using a simple multi-factor clustering approach that classifies each data set using independent observables, thereby assigning each data set to the correct location in conformational space. This procedure is illustrated using two independent observables, unit-cell parameters and intensities, to cluster mixed-state data from chymotrypsinogen (ChTg) crystals. It is observed that the data populate an arc of the reaction trajectory as ChTg is converted into chymotrypsin.

Engineering Protease-Resistant Peptides to Inhibit Human Parainfluenza Viral Respiratory Infection.

The lower respiratory tract infections affecting children worldwide are in large part caused by the parainfluenza viruses (HPIVs), particularly HPIV3, along with human metapneumovirus and respiratory syncytial virus, enveloped negative-strand RNA viruses. There are no vaccines for these important human pathogens, and existing treatments have limited or no efficacy. Infection by HPIV is initiated by viral glycoprotein-mediated fusion between viral and host cell membranes. A viral fusion protein (F), once activated in proximity to a target cell, undergoes a series of conformational changes that first extend the trimer subunits to allow insertion of the hydrophobic domains into the target cell membrane and then refold the trimer into a stable postfusion state, driving the merger of the viral and host cell membranes. Lipopeptides derived from the C-terminal heptad repeat (HRC) domain of HPIV3 F inhibit infection by interfering with the structural transitions of the trimeric F assembly. Clinical application of this strategy, however, requires improving the in vivo stability of antiviral peptides. We show that the HRC peptide backbone can be modified via partial replacement of α-amino acid residues with ß-amino acid residues to generate α/ß-peptides that retain antiviral activity but are poor protease substrates. Relative to a conventional α-lipopeptide, our best α/ß-lipopeptide exhibits improved persistence in vivo and improved anti-HPIV3 antiviral activity in animals.

Effects of Single α-to-ß Residue Replacements on Recognition of an Extended Segment in a Viral Fusion Protein.

Partial replacement of α-amino acid residues with ß-amino acid residues has been established as a strategy for preserving target-engagement by helix-forming polypeptides while altering other properties. The impact of ß-residue incorporation within polypeptides that adopt less regular conformations, however, has received less attention. The C-terminal heptad repeat (HRC) domains of fusion glycoproteins from pathogenic paramyxoviruses contain a segment that must adopt an extended conformation in order to coassemble with the N-terminal heptad repeat (HRN) domain in the postfusion state and drive a merger of the viral envelope with a target cell membrane. Here, we examine the impact of single α-to-ß substitutions within this extended N-terminal segment of an engineered HRC peptide designated VIQKI. Stabilities of hexameric coassemblies formed with the native human parainfluenza virus 3 (HPIV3) HRN have been evaluated, the structures of five coassemblies have been determined, and antiviral efficacies have been measured. Many sites within the extended segment show functional tolerance of α-to-ß substitution. These results offer a basis for future development of paramyxovirus infection inhibitors with novel biological activity profiles, possibly including resistance to proteolysis.

Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic Pathogen Klebsiella oxytoca.

Tilimycin is an enterotoxin produced by the opportunistic pathogen Klebsiella oxytoca that causes antibiotic-associated hemorrhagic colitis (AAHC). This pyrrolobenzodiazepine (PBD) natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway composed of three proteins: NpsA, ThdA, and NpsB. We describe the functional and structural characterization of the fully reconstituted NRPS system and report the steady-state kinetic analysis of all natural substrates and cofactors as well as the structural characterization of both NpsA and ThdA. The mechanism of action of tilimycin was confirmed using DNA adductomics techniques through the detection of putative N-2 guanine alkylation after tilimycin exposure to eukaryotic cells, providing the first structural characterization of a PBD-DNA adduct formed in cells. Finally, we report the rational design of small-molecule inhibitors that block tilimycin biosynthesis in whole cell K. oxytoca (IC50 = 29 ± 4 µM) through the inhibition of NpsA (KD = 29 ± 4 nM).

The structural basis of N-acyl-α-amino-ß-lactone formation catalyzed by a nonribosomal peptide synthetase.

Nonribosomal peptide synthetases produce diverse natural products using a multidomain architecture where the growing peptide, attached to an integrated carrier domain, is delivered to neighboring catalytic domains for bond formation and modification. Investigation of these systems can lead to the discovery of new structures, unusual biosynthetic transformations, and to the engineering of catalysts for generating new products. The antimicrobial ß-lactone obafluorin is produced nonribosomally from dihydroxybenzoic acid and a ß-hydroxy amino acid that cyclizes into the ß-lactone during product release. Here we report the structure of the nonribosomal peptide synthetase ObiF1, highlighting the structure of the ß-lactone-producing thioesterase domain and an interaction between the C-terminal MbtH-like domain with an upstream adenylation domain. Biochemical assays examine catalytic promiscuity, provide mechanistic insight, and demonstrate utility for generating obafluorin analogs. These results advance our understanding of the structural cycle of nonribosomal peptide synthetases and provide insights into the production of ß-lactone natural products.

Dual Inhibition of Human Parainfluenza Type 3 and Respiratory Syncytial Virus Infectivity with a Single Agent.

Human parainfluenza virus 3 (HPIV3) and respiratory syncytial virus (RSV) cause lower respiratory infection in infants and young children. There are no vaccines for these pathogens, and existing treatments have limited or questionable efficacy. Infection by HPIV3 or RSV requires fusion of the viral and cell membranes, a process mediated by a trimeric fusion glycoprotein (F) displayed on the viral envelope. Once triggered, the pre-fusion form of F undergoes a series of conformational changes that first extend the molecule to allow for insertion of the hydrophobic fusion peptide into the target cell membrane and then refold the trimeric assembly into an energetically stable post-fusion state, a process that drives the merger of the viral and host cell membranes. Peptides derived from defined regions of HPIV3 F inhibit infection by HPIV3 by interfering with the structural transitions of the trimeric F assembly. Here we describe lipopeptides derived from the C-terminal heptad repeat (HRC) domain of HPIV3 F that potently inhibit infection by both HPIV3 and RSV. The lead peptide inhibits RSV infection as effectively as does a peptide corresponding to the RSV HRC domain itself. We show that the inhibitors bind to the N-terminal heptad repeat (HRN) domains of both HPIV3 and RSV F with high affinity. Co-crystal structures of inhibitors bound to the HRN domains of HPIV3 or RSV F reveal remarkably different modes of binding in the N-terminal segment of the inhibitor.

Use of a Stereochemical Strategy To Probe the Mechanism of Phenol-Soluble Modulin α3 Toxicity.

Phenol-soluble modulin α3 (PSMα3) is a cytotoxic peptide secreted by virulent strains of Staphylococcus aureus. We used a stereochemical strategy to examine the mechanism of PSMα3-mediated toxicity. One hypothesis is that PSMα3 toxicity requires fibril formation; an alternative is that toxicity is caused by soluble forms of PSMα3, possibly oligomeric. We find that the unnatural enantiomer (D residues) displays cytotoxicity comparable to that of L-PSMα3. Racemic PSMα3 is similarly toxic to enantiopure PSMα3 (L or D) under some conditions, but the toxicity is lost under conditions that cause racemic PSMα3 to aggregate. A crystal structure of racemic PSMα3-NH2 displays an α-helical secondary structure and a packing pattern that is reminiscent of the cross-α arrangement recently discovered in crystals of L-PSMα3. Our data suggest that the cytotoxicity of PSMα3 does not depend on stereospecific engagement of a target protein or other chiral macromolecule, an observation that supports a mechanism based on membrane disruption. In addition, our data support the hypothesis that toxicity is exerted by a soluble form rather than an insoluble fibrillar form.

Retention of Native Quaternary Structure in Racemic Melittin Crystals.

Racemic crystallography has been used to elucidate the secondary and tertiary structures of peptides and small proteins that are recalcitrant to conventional crystallization. It is unclear, however, whether racemic crystallography can capture native quaternary structure, which could be disrupted by heterochiral associations. We are exploring the use of racemic crystallography to characterize the self-assembly behavior of membrane-associated peptides, very few of which have been crystallized. We report a racemic crystal structure of the membrane-active peptide melittin; the new structure allows comparison with a previously reported crystal structure of L-melittin. The tetrameric assembly observed in crystalline L-melittin has been proposed to represent the tetrameric state detected in solution for this peptide. This tetrameric assembly is precisely reproduced in the racemic crystal, which strengthens the conclusion that the tetramer is biologically relevant. More broadly, these findings suggest that racemic crystallography can provide insight on native quaternary structure.

A Hendecad Motif Is Preferred for Heterochiral Coiled-Coil Formation.

The structural principles that govern interactions between l- and d-peptides are not well understood. Among natural proteins, coiled-coil assemblies formed between or among α-helices are the most regular feature of tertiary and quaternary structures. We recently reported the first high-resolution structures for heterochiral coiled-coil dimers, which represent a starting point for understanding associations of l- and d-polypeptides. These structures were an unexpected outcome from crystallization of a racemic peptide corresponding to the transmembrane domain of the influenza A M2 protein (M2-TM). The reported structures raised the possibility that heterochiral coiled-coil dimers prefer an 11-residue (hendecad) sequence repeat, in contrast to the 7-residue (heptad) sequence repeat that is dominant among natural coiled coils. To gain insight on sequence repeat preferences of heterochiral coiled-coils, we have examined three M2-TM variants containing substitutions intended to minimize steric clashes between side chains at the coiled-coil interface. In each of the three new crystal structures, we observed heterochiral coiled-coil associations that closely match a hendecad sequence motif, which strengthens the conclusion that this motif is intrinsic to the pairing of α-helices with opposite handedness. In each case, the presence of a hendecad motif was established by comparing the observed helical frequency to that of an ideal hendecad. This comparison revealed that decreasing the size of the amino acid side chain at positions that project toward the superhelical axis produces tighter packing, as determined by the size of the coiled-coil radius. These results provide a basis for future design of heterochiral coiled-coil pairings.

The Nitro Group as a Masked Electrophile in Covalent Enzyme Inhibition.

We report the unprecedented reaction between a nitroalkane and an active-site cysteine residue to yield a thiohydroximate adduct. Structural and kinetic evidence suggests the nitro group is activated by conversion to its nitronic acid tautomer within the active site. The nitro group, therefore, shows promise as a masked electrophile in the design of covalent inhibitors targeting binding pockets with appropriately placed cysteine and general acid residues.

Evaluation of ß-Amino Acid Replacements in Protein Loops: Effects on Conformational Stability and Structure.

ß-Amino acids have a backbone that is expanded by one carbon atom relative to α-amino acids, and ß residues have been investigated as subunits in protein-like molecules that adopt discrete and predictable conformations. Two classes of ß residue have been widely explored in the context of generating α-helix-like conformations: ß3 -amino acids, which are homologous to α-amino acids and bear a side chain on the backbone carbon adjacent to nitrogen, and residues constrained by a five-membered ring, such the one derived from trans-2-aminocyclopentanecarboxylic acid (ACPC). Substitution of α residues with their ß3  homologues within an α-helix-forming sequence generally causes a decrease in conformational stability. Use of a ring-constrained ß residue, however, can offset the destabilizing effect of αâ†’ß substitution. Here we extend the study of αâ†’ß substitutions, involving both ß3 and ACPC residues, to short loops within a small tertiary motif. We start from previously reported variants of the Pin1 WW domain that contain a two-, three-, or four-residue ß-hairpin loop, and we evaluate αâ†’ß replacements at each loop position for each variant. By referral to the ϕ,ψ angles of the native structure, one can choose a stereochemically appropriate ACPC residue. Use of such logically chosen ACPC residues enhances conformational stability in several cases. Crystal structures of three ß-containing Pin1 WW domain variants show that a native-like tertiary structure is maintained in each case.