Accurate de novo design of membrane-traversing macrocycles.

We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed 184 6-12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6-12 residue size range cross membranes with an apparent permeability greater than 1 × 10-6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.

De novo protein design by deep network hallucination.

There has been considerable recent progress in protein structure prediction using deep neural networks to predict inter-residue distances from amino acid sequences1-3. Here we investigate whether the information captured by such networks is sufficiently rich to generate new folded proteins with sequences unrelated to those of the naturally occurring proteins used in training the models. We generate random amino acid sequences, and input them into the trRosetta structure prediction network to predict starting residue-residue distance maps, which, as expected, are quite featureless. We then carry out Monte Carlo sampling in amino acid sequence space, optimizing the contrast (Kullback-Leibler divergence) between the inter-residue distance distributions predicted by the network and background distributions averaged over all proteins. Optimization from different random starting points resulted in novel proteins spanning a wide range of sequences and predicted structures. We obtained synthetic genes encoding 129 of the network-'hallucinated' sequences, and expressed and purified the proteins in Escherichia coli; 27 of the proteins yielded monodisperse species with circular dichroism spectra consistent with the hallucinated structures. We determined the three-dimensional structures of three of the hallucinated proteins, two by X-ray crystallography and one by NMR, and these closely matched the hallucinated models. Thus, deep networks trained to predict native protein structures from their sequences can be inverted to design new proteins, and such networks and methods should contribute alongside traditional physics-based models to the de novo design of proteins with new functions.

Enumerative Discovery of Noncanonical Polypeptide Secondary Structures.

Energetically favorable local interactions can overcome the entropic cost of chain ordering and cause otherwise flexible polymers to adopt regularly repeating backbone conformations. A prominent example is the α helix present in many protein structures, which is stabilized by i, i + 4 hydrogen bonds between backbone peptide units. With the increased chemical diversity offered by unnatural amino acids and backbones, it has been possible to identify regularly repeating structures not present in proteins, but to date, there has been no systematic approach for identifying new polymers likely to have such structures despite their considerable potential for molecular engineering. Here we describe a systematic approach to search through dipeptide combinations of 130 chemically diverse amino acids to identify those predicted to populate unique low-energy states. We characterize ten newly identified dipeptide repeating structures using circular dichroism spectroscopy and comparison with calculated spectra. NMR and X-ray crystallographic structures of two of these dipeptide-repeat polymers are similar to the computational models. Our approach is readily generalizable to identify low-energy repeating structures for a wide variety of polymers, and our ordered dipeptide repeats provide new building blocks for molecular engineering.

Structural asymmetry in the closed state of mitochondrial Hsp90 (TRAP1) supports a two-step ATP hydrolysis mechanism.

While structural symmetry is a prevailing feature of homo-oligomeric proteins, asymmetry provides unique mechanistic opportunities. We present the crystal structure of full-length TRAP1, the mitochondrial Hsp90 molecular chaperone, in a catalytically active closed state. The TRAP1 homodimer adopts a distinct, asymmetric conformation, where one protomer is reconfigured via a helix swap at the middle:C-terminal domain (MD:CTD) interface. This interface plays a critical role in client binding. Solution methods validate the asymmetry and show extension to Hsp90 homologs. Point mutations that disrupt unique contacts at each MD:CTD interface reduce catalytic activity and substrate binding and demonstrate that each protomer needs access to both conformations. Crystallographic data on a dimeric NTD:MD fragment suggests that asymmetry arises from strain induced by simultaneous NTD and CTD dimerization. The observed asymmetry provides the potential for an additional step in the ATPase cycle, allowing sequential ATP hydrolysis steps to drive both client remodeling and client release.

Structural insight into the length-dependent binding of ssDNA by SP_0782 from Streptococcus pneumoniae, reveals a divergence in the DNA-binding interface of PC4-like proteins.

SP_0782 from Streptococcus pneumoniae is a dimeric protein that potentially binds with single-stranded DNA (ssDNA) in a manner similar to human PC4, the prototype of PC4-like proteins, which plays roles in transcription and maintenance of genome stability. In a previous NMR study, SP_0782 exhibited an ssDNA-binding property different from YdbC, a prokaryotic PC4-like protein from Lactococcus lactis, but the underlying mechanism remains unclear. Here, we show that although SP_0782 adopts an overall fold similar to those of PC4 and YdbC, the ssDNA length occupied by SP_0782 is shorter than those occupied by PC4 and YdbC. SP_0782 exhibits varied binding patterns for different lengths of ssDNA, and tends to form large complexes with ssDNA in a potential high-density binding manner. The structures of SP_0782 complexed with different ssDNAs reveal that the varied binding patterns are associated with distinct capture of nucleotides in two major DNA-binding regions of SP_0782. Moreover, a comparison of known structures of PC4-like proteins complexed with ssDNA reveals a divergence in the binding interface between prokaryotic and eukaryotic PC4-like proteins. This study provides insights into the ssDNA-binding mechanism of PC4-like proteins, and benefits further study regarding the biological function of SP_0782, probably in DNA protection and natural transformation.

Solution NMR structure of CGL2373, a polyketide cyclase-like protein from Corynebacterium glutamicum.

Protein CGL2373 from Corynebacterium glutamicum was previously proposed to be a member of the polyketide_cyc2 family, based on amino-acid sequence and secondary structure features derived from NMR chemical shift assignments. We report here the solution NMR structure of CGL2373, which contains three α-helices and one antiparallel ß-sheet and adopts a helix-grip fold. This structure shows moderate similarities to the representative polyketide cyclases, TcmN, WhiE, and ZhuI. Nevertheless, unlike the structures of these homologs, CGL2373 structure looks like a half-open shell with a much larger pocket, and key residues in the representative polyketide cyclases for binding substrate and catalyzing aromatic ring formation are replaced with different residues in CGL2373. Also, the gene cluster where the CGL2373-encoding gene is located in C. glutamicum contains additional genes encoding nucleoside diphosphate kinase, folylpolyglutamate synthase, and valine-tRNA ligase, different from the typical gene cluster encoding polyketide cyclase in Streptomyces. Thus, although CGL2373 is structurally a polyketide cyclase-like protein, the function of CGL2373 may differ from the known polyketide cyclases and needs to be further investigated. The solution structure of CGL2373 lays a foundation for in silico ligand screening and binding site identifying in future functional study.

Solution NMR structure of CHU_1110 from Cytophaga hutchinsonii, an AHSA1 protein potentially involved in metal ion stress response.

We report the solution nuclear magnetic resonance (NMR) structure of CHU_1110 from Cytophaga hutchinsonii. CHU_1110 contains three α-helices and one antiparallel ß-sheet, forming a large cavity in the center of the protein, which are consistent with the structural characteristics of AHSA1 protein family. This protein shows high structural similarities to the prokaryotic proteins RHE_CH02687 from Rhizobium etli and YndB from Bacillus subtilis, which can bind with flavinoids. Unlike these two homologs, CHU_1110 shows no obvious interaction with flavonoids in NMR titration experiments. In addition, no direct interaction has been observed between CHU_1110 and ATP, although many homologous sequences of CHU_1110 have been annotated as ATPase. Combining the analysis of structural similarity of CHU_1110 and genomic context of its encoding gene, we speculate that CHU_1110 may be involved in the stress response of bacteria to heavy metal ions, even though its specific biological functions that need to be further investigated.

Solution NMR structure and ligand identification of human Gas7 SH3 domain reveal a typical SH3 fold but a non-canonical ligand-binding mode.

Growth arrest specific 7 (Gas7) protein is a cytoskeleton regulator playing a crucial role in neural cell development and function, and has been implicated in Alzheimer disease, schizophrenia and cancers. In human, three Gas7 isoforms can be expressed from a single Gas7 gene, while only the longest isoform, hGas7c, possesses an SH3 domain at the N-terminus. To date, the structure and function of hGas7 SH3 domain are still unclear. Here, we reported the solution NMR structure of hGas7 SH3 domain (hGas7-SH3), which displays a typical SH3 ß-barrel fold comprising five ß-strands and one 310-helix. Structural and sequence comparison showed that hGas7-SH3 shares high similarity with Abl SH3 domain, which binds to a high-affinity proline-rich peptide P41 in a canonical SH3-ligand binding mode through two hydrophobic pockets and a specificity site in the RT-loop. However, unlike Abl-SH3, only six residues in the RT-loop and two residues adjacent to but not in the two hydrophobic pockets of hGas7-SH3 showed significant chemical shift perturbations in NMR titrations, suggesting a low affinity and a non-canonical binding mode of hGas7-SH3 for P41. Furthermore, four peptides selected from phage-displayed libraries also bound weakly to hGas7-SH3, and the binding region of hGas7-SH3 was mainly located in the RT-loop as well. The ligand identifications through structural similarity searching and peptide library screening in this study imply that although hGas7-SH3 adopts a typical SH3 fold, it probably possesses distinctive ligand-binding specificity.

In vivo production of psilocybin in E. coli.

Psilocybin, the prodrug of the psychoactive molecule psilocin, has demonstrated promising results in clinical trials for the treatment of addiction, depression, and post-traumatic stress disorder. The development of a psilocybin production platform in a highly engineerable microbe could lead to rapid advances towards the bioproduction of psilocybin for use in ongoing clinical trials. Here, we present the development of a modular biosynthetic production platform in the model microbe, Escherichia coli. Efforts to optimize and improve pathway performance using multiple genetic optimization techniques were evaluated, resulting in a 32-fold improvement in psilocybin titer. Further enhancements to this genetically superior strain were achieved through fermentation optimization, ultimately resulting in a fed-batch fermentation study, with a production titer of 1.16 g/L of psilocybin. This is the highest psilocybin titer achieved to date from a recombinant organism and a significant step towards demonstrating the feasibility of industrial production of biologically-derived psilocybin.

Structural insights into the impact of two holoprosencephaly-related mutations on human TGIF1 homeodomain.

Human protein TGIF1 is an essential regulator of cell fate with broad roles in different tissues, and has been implicated in holoprosencephaly (HPE) and many cancers. The function of TGIF1 in transcriptional regulation depends on its three-amino acid loop extension (TALE) type of homeodomain (HD). Two missense mutations that led to P192A and R219C substitutions in TGIF1-HD were previously found in HPE patients and suggested to be the causes for these cases. However, how these mutations affected TGIF1 function has not been investigated from a structural view. Here, we investigated the roles of P192 and R219 in TGIF1-HD structure packing through determining the NMR structure of TGIF1-HD. Surprisingly, P192 and R219 were found to play roles in packing α1 and α2 to α3 together with A190 and F215 through side-chain interactions. Circular dichroism (CD) showed that P192A and R219C mutants displayed structural change and less folding compared with wild-type TGIF1-HD, and 1H-15N HSQC spectrum of P192A mutant exhibited chemical shift perturbations in all three helices of TGIF1-HD. Thus, it is suggested that P192A and R219C mutations led to structure disturbances of TGIF1-HD, which subsequently reduced the DNA-binding affinity of TGIF1-HD by 23-fold and 10-fold respectively, as revealed by the isothermal titration calorimetry (ITC) experiments. Our study provides structural insights of the probable pathogenesis mechanism of two TGIF1-related HPE cases, and evidences for the roles of P192 and R219 in HD folding.

Solution structure of SHIP2 SH2 domain and its interaction with a phosphotyrosine peptide from c-MET.

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) binds with the Y1356-phosphorylated hepatocyte growth factor (HGF) receptor, c-MET, through its SH2 domain, which is essential for the role of SHIP2 in HGF-induced cell scattering and cell spreading. Previously, the experimental structure of the SH2 domain from SHIP2 (SHIP2-SH2) had not been reported, and its interaction with the Y1356-phosphorylated c-MET had not been investigated from a structural point of view. In this study, the solution structure of SHIP2-SH2 was determined by NMR spectroscopy, where it was found to adopt a typical SH2-domain fold that contains a positively-charged pocket for binding to phosphotyrosine (pY). The interaction between SHIP2-SH2 and a pY-containing peptide from c-MET (Y1356 phosphorylated) was investigated through NMR titrations. The results showed that the binding affinity of SHIP2-SH2 with the phosphopeptide is at low micromolar level, and the binding interface consists of the positively-charged pocket and its surrounding regions. Furthermore, R28, S49 and R70 were identified as key residues for the binding and may directly interact with the pY. Taken together, these findings provide structural insights into the binding of SHIP2-SH2 with the Y1356-phosphorylated c-MET, and lay a foundation for further studies of the interactions between SHIP2-SH2 and its various binding partners.

Solution NMR structure of zinc finger 4 and 5 from human INSM1, an essential regulator of neuroendocrine differentiation.

Human INSM1 containing five C-terminal C2H2-type zinc fingers (ZFs), is a key regulator of neuroendocrine development. Previous research reported that full-length INSM1 containing all five ZFs recognized a consensus DNA sequence. Structure elucidation of human INSM1 ZFs is currently insufficient to understand the DNA binding mechanism. Herein, we present the solution NMR structure of ZF4-5, in which the two ZFs adopt a head-to-tail arrangement and each ZF features a canonical ßßα fold. NMR titrations and isothermal titration calorimetry experiments showed that ZF4-5 binds weakly to the consensus DNA sequence. Proteins 2017; 85:957-962. © 2016 Wiley Periodicals, Inc.

Solution NMR structure of RHE_CH02687 from Rhizobium etli: A novel flavonoid-binding protein.

We report the solution NMR structure of RHE_CH02687 from Rhizobium etli. Its structure consists of two ß-sheets that together with two short and one long α-helix form a hydrophobic cavity. This protein shows a high structural similarity to the prokaryotic protein YndB from Bacillus subtilis, and the eukaryotic protein Aha1. NMR titration experiments confirmed that RHE_CH02687, like its homolog YndB, interacted with flavonoids, giving support for a biological function as a flavonoid sensor in the symbiotic interaction between R. etli and plants. In addition, our study showed no evidence for a direct interaction between RHE_CH02687 and HtpG, the R. etli homolog of Hsp90. Proteins 2017; 85:951-956. © 2016 Wiley Periodicals, Inc.

NMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with 13C-methyl alanine.

A strategy for acquiring structural information from sparsely isotopically labeled large proteins is illustrated with an application to the E. coli heat-shock protein, HtpG (high temperature protein G), a 145 kDa dimer. It uses 13C-alanine methyl labeling in a perdeuterated background to take advantage of the sensitivity and resolution of Methyl-TROSY spectra, as well as the backbone-centered structural information from 1H-13C residual dipolar couplings (RDCs) of alanine methyl groups. In all, 40 of the 47 expected crosspeaks were resolved and 36 gave RDC data. Assignments of crosspeaks were partially achieved by transferring assignments from those made on individual domains using triple resonance methods. However, these were incomplete and in many cases the transfer was ambiguous. A genetic algorithm search for consistency between predictions based on domain structures and measurements for chemical shifts and RDCs allowed 60% of the 40 resolved crosspeaks to be assigned with confidence. Chemical shift changes of these crosspeaks on adding an ATP analog to the apo-protein are shown to be consistent with structural changes expected on comparing previous crystal structures for apo- and complex- structures. RDCs collected on the assigned alanine methyl peaks are used to generate a new solution model for the apo-protein structure.

Glycan Specificity of P[19] Rotavirus and Comparison with Those of Related P Genotypes.

The P[19] genotype belongs to the P[II] genogroup of group A rotaviruses (RVs). However, unlike the other P[II] RVs, which mainly infect humans, P[19] RVs commonly infect animals (pigs), making P[19] unique for the study of RV diversity and host ranges. Through in vitro binding assays and saturation transfer difference (STD) nuclear magnetic resonance (NMR), we found that P[19] could bind mucin cores 2, 4, and 6, as well as type 1 histo-blood group antigens (HBGAs). The common sequences of these glycans serve as minimal binding units, while additional residues, such as the A, B, H, and Lewis epitopes of the type 1 HBGAs, can further define the binding outcomes and therefore likely the host ranges for P[19] RVs. This complex binding property of P[19] is shared with the other three P[II] RVs (P[4], P[6], and P[8]) in that all of them recognized the type 1 HBGA precursor, although P[4] and P[8], but not P[6], also bind to mucin cores. Moreover, while essential for P[4] and P[8] binding, the addition of the Lewis epitope blocked P[6] and P[19] binding to type 1 HBGAs. Chemical-shift NMR of P[19] VP8* identified a ligand binding interface that has shifted away from the known RV P-genotype binding sites but is conserved among all P[II] RVs and two P[I] RVs (P[10] and P[12]), suggesting an evolutionary connection among these human and animal RVs. Taken together, these data are important for hypotheses on potential mechanisms for RV diversity, host ranges, and cross-species transmission. IMPORTANCE: In this study, we found that our P[19] strain and other P[II] RVs recognize mucin cores and the type 1 HBGA precursors as the minimal functional units and that additional saccharides adjacent to these units can alter binding outcomes and thereby possibly host ranges. These data may help to explain why some P[II] RVs, such as P[6] and P[19], commonly infect animals but rarely humans, while others, such as the P[4] and P[8] RVs, mainly infect humans and are predominant over other P genotypes. Elucidation of the molecular bases for strain-specific host ranges and cross-species transmission of these human and animal RVs is important to understand RV epidemiology and disease burden, which may impact development of control and prevention strategies against RV gastroenteritis.

Tumor-derived RHOA mutants interact with effectors in the GDP-bound state.

RHOA mutations are found at diverse residues in various cancer types, implying mutation- and cell-specific mechanisms of tumorigenesis. Here, we focus on the underlying mechanisms of two gain-of-function RHOA mutations, A161P and A161V, identified in adult T-cell leukemia/lymphoma. We find that RHOAA161P and RHOAA161V are both fast-cycling mutants with increased guanine nucleotide dissociation/association rates compared with RHOAWT and show reduced GTP-hydrolysis activity. Crystal structures reveal an altered nucleotide association in RHOAA161P and an open nucleotide pocket in RHOAA161V. Both mutations perturb the dynamic properties of RHOA switch regions and shift the conformational landscape important for RHOA activity, as shown by 31P NMR and molecular dynamics simulations. Interestingly, RHOAA161P and RHOAA161V can interact with effectors in the GDP-bound state. 1H-15N HSQC NMR spectra support the existence of an active population in RHOAA161V-GDP. The distinct interaction mechanisms resulting from the mutations likely favor an RHOAWT-like "ON" conformation, endowing GDP-bound state effector binding activity.

The 100-protein NMR spectra dataset: A resource for biomolecular NMR data analysis.

Multidimensional NMR spectra are the basis for studying proteins by NMR spectroscopy and crucial for the development and evaluation of methods for biomolecular NMR data analysis. Nevertheless, in contrast to derived data such as chemical shift assignments in the BMRB and protein structures in the PDB databases, this primary data is in general not publicly archived. To change this unsatisfactory situation, we present a standardized set of solution NMR data comprising 1329 2-4-dimensional NMR spectra and associated reference (chemical shift assignments, structures) and derived (peak lists, restraints for structure calculation, etc.) annotations. With the 100-protein NMR spectra dataset that was originally compiled for the development of the ARTINA deep learning-based spectra analysis method, 100 protein structures can be reproduced from their original experimental data. The 100-protein NMR spectra dataset is expected to help the development of computational methods for NMR spectroscopy, in particular machine learning approaches, and enable consistent and objective comparisons of these methods.

Sequence-specific targeting of intrinsically disordered protein regions.

A general approach to design proteins that bind tightly and specifically to intrinsically disordered regions (IDRs) of proteins and flexible peptides would have wide application in biological research, therapeutics, and diagnosis. However, the lack of defined structures and the high variability in sequence and conformational preferences has complicated such efforts. We sought to develop a method combining biophysical principles with deep learning to readily generate binders for any disordered sequence. Instead of assuming a fixed regular structure for the target, general recognition is achieved by threading the query sequence through diverse extended binding modes in hundreds of templates with varying pocket depths and spacings, followed by RFdiffusion refinement to optimize the binder-target fit. We tested the method by designing binders to 39 highly diverse unstructured targets. Experimental testing of ~36 designs per target yielded binders with affinities better than 100 nM in 34 cases, and in the pM range in four cases. The co-crystal structure of a designed binder in complex with dynorphin A is closely consistent with the design model. All by all binding experiments for 20 designs binding diverse targets show they are highly specific for the intended targets, with no crosstalk even for the closely related dynorphin A and dynorphin B. Our approach thus could provide a general solution to the intrinsically disordered protein and peptide recognition problem.

Restraint Validation of Biomolecular Structures Determined by NMR in the Protein Data Bank.

Biomolecular structure analysis from experimental NMR studies generally relies on restraints derived from a combination of experimental and knowledge-based data. A challenge for the structural biology community has been a lack of standards for representing these restraints, preventing the establishment of uniform methods of model-vs-data structure validation against restraints and limiting interoperability between restraint-based structure modeling programs. The NMR exchange (NEF) and NMR-STAR formats provide a standardized approach for representing commonly used NMR restraints. Using these restraint formats, a standardized validation system for assessing structural models of biopolymers against restraints has been developed and implemented in the wwPDB OneDep data deposition-validation-biocuration system. The resulting wwPDB Restraint Violation Report provides a model vs. data assessment of biomolecule structures determined using distance and dihedral restraints, with extensions to other restraint types currently being implemented. These tools are useful for assessing NMR models, as well as for assessing biomolecular structure predictions based on distance restraints.

Restraint validation of biomolecular structures determined by NMR in the Protein Data Bank.

Biomolecular structure analysis from experimental NMR studies generally relies on restraints derived from a combination of experimental and knowledge-based data. A challenge for the structural biology community has been a lack of standards for representing these restraints, preventing the establishment of uniform methods of model-vs-data structure validation against restraints and limiting interoperability between restraint-based structure modeling programs. The NEF and NMR-STAR formats provide a standardized approach for representing commonly used NMR restraints. Using these restraint formats, a standardized validation system for assessing structural models of biopolymers against restraints has been developed and implemented in the wwPDB OneDep data deposition-validation-biocuration system. The resulting wwPDB restraint violation report provides a model vs. data assessment of biomolecule structures determined using distance and dihedral restraints, with extensions to other restraint types currently being implemented. These tools are useful for assessing NMR models, as well as for assessing biomolecular structure predictions based on distance restraints.