Codon influence on protein expression in E. coli correlates with mRNA levels.

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli.

Principles for designing ideal protein structures.

Unlike random heteropolymers, natural proteins fold into unique ordered structures. Understanding how these are encoded in amino-acid sequences is complicated by energetically unfavourable non-ideal features--for example kinked α-helices, bulged ß-strands, strained loops and buried polar groups--that arise in proteins from evolutionary selection for biological function or from neutral drift. Here we describe an approach to designing ideal protein structures stabilized by completely consistent local and non-local interactions. The approach is based on a set of rules relating secondary structure patterns to protein tertiary motifs, which make possible the design of funnel-shaped protein folding energy landscapes leading into the target folded state. Guided by these rules, we designed sequences predicted to fold into ideal protein structures consisting of α-helices, ß-strands and minimal loops. Designs for five different topologies were found to be monomeric and very stable and to adopt structures in solution nearly identical to the computational models. These results illuminate how the folding funnels of natural proteins arise and provide the foundation for engineering a new generation of functional proteins free from natural evolution.

X-ray crystal structure of the N-terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition.

The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N-terminal domain (NTD), the catalytic core domain, and the C-terminal domain. The NTD includes an HHCC zinc finger-like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M-MuLV) IN N-terminal region (NTR) constructs that both include an N-terminal extension domain (NED, residues 1-44) and an HHCC zinc-finger NTD (residues 45-105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1-105 (NTR1-105 ) and 8-105 (NTR8-105 ) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR1-105 packs as a dimer and NTR8-105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti-parallel ß-strands and an α-helix, similar to the NED of prototype foamy virus (PFV) IN. These three ß-strands form an extended ß-sheet with another ß-strand in the HHCC Zn2+ binding domain, which is a unique structural feature for the M-MuLV IN. The HHCC Zn2+ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M-MuLV. Proteins 2017; 85:647-656. © 2016 Wiley Periodicals, Inc.

A hybrid NMR/SAXS-based approach for discriminating oligomeric protein interfaces using Rosetta.

Oligomeric proteins are important targets for structure determination in solution. While in most cases the fold of individual subunits can be determined experimentally, or predicted by homology-based methods, protein-protein interfaces are challenging to determine de novo using conventional NMR structure determination protocols. Here we focus on a member of the bet-V1 superfamily, Aha1 from Colwellia psychrerythraea. This family displays a broad range of crystallographic interfaces none of which can be reconciled with the NMR and SAXS data collected for Aha1. Unlike conventional methods relying on a dense network of experimental restraints, the sparse data are used to limit conformational search during optimization of a physically realistic energy function. This work highlights a new approach for studying minor conformational changes due to structural plasticity within a single dimeric interface in solution.

The second round of Critical Assessment of Automated Structure Determination of Proteins by NMR: CASD-NMR-2013.

The second round of the community-wide initiative Critical Assessment of automated Structure Determination of Proteins by NMR (CASD-NMR-2013) comprised ten blind target datasets, consisting of unprocessed spectral data, assigned chemical shift lists and unassigned NOESY peak and RDC lists, that were made available in both curated (i.e. manually refined) or un-curated (i.e. automatically generated) form. Ten structure calculation programs, using fully automated protocols only, generated a total of 164 three-dimensional structures (entries) for the ten targets, sometimes using both curated and un-curated lists to generate multiple entries for a single target. The accuracy of the entries could be established by comparing them to the corresponding manually solved structure of each target, which was not available at the time the data were provided. Across the entire data set, 71 % of all entries submitted achieved an accuracy relative to the reference NMR structure better than 1.5 Å. Methods based on NOESY peak lists achieved even better results with up to 100% of the entries within the 1.5 Å threshold for some programs. However, some methods did not converge for some targets using un-curated NOESY peak lists. Over 90% of the entries achieved an accuracy better than the more relaxed threshold of 2.5 Å that was used in the previous CASD-NMR-2010 round. Comparisons between entries generated with un-curated versus curated peaks show only marginal improvements for the latter in those cases where both calculations converged.

Two Fe-S clusters catalyze sulfur insertion by radical-SAM methylthiotransferases.

How living organisms create carbon-sulfur bonds during the biosynthesis of critical sulfur-containing compounds is still poorly understood. The methylthiotransferases MiaB and RimO catalyze sulfur insertion into tRNAs and ribosomal protein S12, respectively. Both belong to a subgroup of radical-S-adenosylmethionine (radical-SAM) enzymes that bear two [4Fe-4S] clusters. One cluster binds S-adenosylmethionine and generates an Ado• radical via a well-established mechanism. However, the precise role of the second cluster is unclear. For some sulfur-inserting radical-SAM enzymes, this cluster has been proposed to act as a sacrificial source of sulfur for the reaction. In this paper, we report parallel enzymological, spectroscopic and crystallographic investigations of RimO and MiaB, which provide what is to our knowledge the first evidence that these enzymes are true catalysts and support a new sulfation mechanism involving activation of an exogenous sulfur cosubstrate at an exchangeable coordination site on the second cluster, which remains intact during the reaction.

Structures of apo- and ssDNA-bound YdbC from Lactococcus lactis uncover the function of protein domain family DUF2128 and expand the single-stranded DNA-binding domain proteome.

Single-stranded DNA (ssDNA) binding proteins are important in basal metabolic pathways for gene transcription, recombination, DNA repair and replication in all domains of life. Their main cellular role is to stabilize melted duplex DNA and protect genomic DNA from degradation. We have uncovered the molecular function of protein domain family domain of unknown function DUF2128 (PF09901) as a novel ssDNA binding domain. This bacterial domain strongly associates into a dimer and presents a highly positively charged surface that is consistent with its function in non-specific ssDNA binding. Lactococcus lactis YdbC is a representative of DUF2128. The solution NMR structures of the 20 kDa apo-YdbC dimer and YdbC:dT(19)G(1) complex were determined. The ssDNA-binding energetics to YdbC were characterized by isothermal titration calorimetry. YdbC shows comparable nanomolar affinities for pyrimidine and mixed oligonucleotides, and the affinity is sufficiently strong to disrupt duplex DNA. In addition, YdbC binds with lower affinity to ssRNA, making it a versatile nucleic acid-binding domain. The DUF2128 family is related to the eukaryotic nuclear protein positive cofactor 4 (PC4) family and to the PUR family both by fold similarity and molecular function.

Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples.

We have developed an approach for determining NMR structures of proteins over 20 kDa that utilizes sparse distance restraints obtained using transverse relaxation optimized spectroscopy experiments on perdeuterated samples to guide RASREC Rosetta NMR structure calculations. The method was tested on 11 proteins ranging from 15 to 40 kDa, seven of which were previously unsolved. The RASREC Rosetta models were in good agreement with models obtained using traditional NMR methods with larger restraint sets. In five cases X-ray structures were determined or were available, allowing comparison of the accuracy of the Rosetta models and conventional NMR models. In all five cases, the Rosetta models were more similar to the X-ray structures over both the backbone and side-chain conformations than the "best effort" structures determined by conventional methods. The incorporation of sparse distance restraints into RASREC Rosetta allows routine determination of high-quality solution NMR structures for proteins up to 40 kDa, and should be broadly useful in structural biology.

Solution NMR structures of homeodomains from human proteins ALX4, ZHX1, and CASP8AP2 contribute to the structural coverage of the Human Cancer Protein Interaction Network.

High-quality solution NMR structures of three homeodomains from human proteins ALX4, ZHX1 and CASP8AP2 were solved. These domains were chosen as targets of a biomedical theme project pursued by the Northeast Structural Genomics Consortium. This project focuses on increasing the structural coverage of human proteins associated with cancer.

Solution NMR structures of immunoglobulin-like domains 7 and 12 from obscurin-like protein 1 contribute to the structural coverage of the Human Cancer Protein Interaction Network.

High-quality solution NMR structures of immunoglobulin-like domains 7 and 12 from human obscurin-like protein 1 were solved. The two domains share 30% sequence identity and their structures are, as expected, rather similar. The new structures contribute to structural coverage of human cancer associated proteins. Mutations of Arg 812 in domain 7 cause the rare 3-M syndrome, and this site is located in a surface area predicted to be involved in protein-protein interactions.

Structure and function of human DnaJ homologue subfamily a member 1 (DNAJA1) and its relationship to pancreatic cancer.

Pancreatic cancer has a dismal 5 year survival rate of 5.5% that has not been improved over the past 25 years despite an enormous amount of effort. Thus, there is an urgent need to identify truly novel yet druggable protein targets for drug discovery. The human protein DnaJ homologue subfamily A member 1 (DNAJA1) was previously shown to be downregulated 5-fold in pancreatic cancer cells and has been targeted as a biomarker for pancreatic cancer, but little is known about the specific biological function for DNAJA1 or the other members of the DnaJ family encoded in the human genome. Our results suggest the overexpression of DNAJA1 suppresses the stress response capabilities of the oncogenic transcription factor, c-Jun, and results in the diminution of cell survival. DNAJA1 likely activates a DnaK protein by forming a complex that suppresses the JNK pathway, the hyperphosphorylation of c-Jun, and the anti-apoptosis state found in pancreatic cancer cells. A high-quality nuclear magnetic resonance solution structure of the J-domain of DNAJA1 combined with a bioinformatics analysis and a ligand affinity screen identifies a potential DnaK binding site, which is also predicted to overlap with an inhibitory binding site, suggesting DNAJA1 activity is highly regulated.

Solution NMR structure of the helicase associated domain BVU_0683(627-691) from Bacteroides vulgatus provides first structural coverage for protein domain family PF03457 and indicates domain binding to DNA.

A high-quality NMR structure of the helicase associated (HA) domain comprising residues 627-691 of the 753-residue protein BVU_0683 from Bacteroides vulgatus exhibits an all α-helical fold. The structure presented here is the first representative for the large protein domain family PF03457 (currently 742 members) of HA domains. Comparison with structurally similar proteins supports the hypothesis that HA domains bind to DNA and that binding specificity varies greatly within the family of HA domains constituting PF03457.

Solution NMR structure of CD1104B from pathogenic Clostridium difficile reveals a distinct α-helical architecture and provides first structural representative of protein domain family PF14203.

A high-quality structure of the 68-residue protein CD1104B from Clostridium difficile strain 630 exhibits a distinct all α-helical fold. The structure presented here is the first representative of bacterial protein domain family PF14203 (currently 180 members) of unknown function (DUF4319) and reveals that the side-chains of the only two strictly conserved residues (Glu 8 and Lys 48) form a salt bridge. Moreover, these two residues are located in the vicinity of the largest surface cleft which is predicted to contribute to a surface area involved in protein-protein interactions. This, along with its coding in transposon CTn4, suggests that CD1104B (and very likely all members of Pfam 14203) functions by interacting with other proteins required for the transfer of transposons between different bacterial species.

Solution NMR structures provide first structural coverage of the large protein domain family PF08369 and complementary structural coverage of dark operative protochlorophyllide oxidoreductase complexes.

High-quality NMR structures of the C-terminal domain comprising residues 484-537 of the 537-residue protein Bacterial chlorophyll subunit B (BchB) from Chlorobium tepidum and residues 9-61 of 61-residue Asr4154 from Nostoc sp. (strain PCC 7120) exhibit a mixed α/ß fold comprised of three α-helices and a small ß-sheet packed against second α-helix. These two proteins share 29% sequence similarity and their structures are globally quite similar. The structures of BchB(484-537) and Asr4154(9-61) are the first representative structures for the large protein family (Pfam) PF08369, a family of unknown function currently containing 610 members in bacteria and eukaryotes. Furthermore, BchB(484-537) complements the structural coverage of the dark-operating protochlorophyllide oxidoreductase.

Structural and biochemical characterization of the bilin lyase CpcS from Thermosynechococcus elongatus.

Cyanobacterial phycobiliproteins have evolved to capture light energy over most of the visible spectrum due to their bilin chromophores, which are linear tetrapyrroles that have been covalently attached by enzymes called bilin lyases. We report here the crystal structure of a bilin lyase of the CpcS family from Thermosynechococcus elongatus (TeCpcS-III). TeCpcS-III is a 10-stranded ß barrel with two alpha helices and belongs to the lipocalin structural family. TeCpcS-III catalyzes both cognate as well as noncognate bilin attachment to a variety of phycobiliprotein subunits. TeCpcS-III ligates phycocyanobilin, phycoerythrobilin, and phytochromobilin to the alpha and beta subunits of allophycocyanin and to the beta subunit of phycocyanin at the Cys82-equivalent position in all cases. The active form of TeCpcS-III is a dimer, which is consistent with the structure observed in the crystal. With the use of the UnaG protein and its association with bilirubin as a guide, a model for the association between the native substrate, phycocyanobilin, and TeCpcS was produced.

Protocol for production and purification of SARS-CoV-2 3CLpro.

3CLpro protease from SARS-CoV-2 is a primary target for COVID-19 antiviral drug development. Here, we present a protocol for 3CLpro production in Escherichia coli. We describe steps to purify 3CLpro, expressed as a fusion with the Saccharomyces cerevisiae SUMO protein, with yields up to 120 mg L-1 following cleavage. The protocol also provides isotope-enriched samples suitable for nuclear magnetic resonance (NMR) studies. We also present methods to characterize 3CLpro by mass spectrometry, X-ray crystallography, heteronuclear NMR, and a Förster-resonance-energy-transfer-based enzyme assay. For complete details on the use and execution of this protocol, please refer to Bafna et al.1.

Solution NMR structures reveal a distinct architecture and provide first structures for protein domain family PF04536.

The protein family (Pfam) PF04536 is a broadly conserved domain family of unknown function (DUF477), with more than 1,350 members in prokaryotic and eukaryotic proteins. High-quality NMR structures of the N-terminal domain comprising residues 41-180 of the 684-residue protein CG2496 from Corynebacterium glutamicum and the N-terminal domain comprising residues 35-182 of the 435-residue protein PG0361 from Porphyromonas gingivalis both exhibit an α/ß fold comprised of a four-stranded ß-sheet, three α-helices packed against one side of the sheet, and a fourth α-helix attached to the other side. In spite of low sequence similarity (18%) assessed by structure-based sequence alignment, the two structures are globally quite similar. However, moderate structural differences are observed for the relative orientation of two of the four helices. Comparison with known protein structures reveals that the α/ß architecture of CG2496(41-180) and PG0361(35-182) has previously not been characterized. Moreover, calculation of surface charge potential and identification of surface clefts indicate that the two domains very likely have different functions.

Crystal structure of a catalytically active GG(D/E)EF diguanylate cyclase domain from Marinobacter aquaeolei with bound c-di-GMP product.

Recent studies of signal transduction in bacteria have revealed a unique second messenger, bis-(3'-5')-cyclic dimeric GMP (c-di-GMP), which regulates transitions between motile states and sessile states, such as biofilms. C-di-GMP is synthesized from two GTP molecules by diguanylate cyclases (DGC). The catalytic activity of DGCs depends on a conserved GG(D/E)EF domain, usually part of a larger multi-domain protein organization. The domains other than the GG(D/E)EF domain often control DGC activation. This paper presents the 1.83 Å crystal structure of an isolated catalytically competent GG(D/E)EF domain from the A1U3W3_MARAV protein from Marinobacter aquaeolei. Co-crystallization with GTP resulted in enzymatic synthesis of c-di-GMP. Comparison with previously solved DGC structures shows a similar orientation of c-di-GMP bound to an allosteric regulatory site mediating feedback inhibition of the enzyme. Biosynthesis of c-di-GMP in the crystallization reaction establishes that the enzymatic activity of this DGC domain does not require interaction with regulatory domains.

Solution NMR structure of Alr2454 from Nostoc sp. PCC 7120, the first structural representative of Pfam domain family PF11267.

Protein domain family PF11267 (DUF3067) is a family of proteins of unknown function found in both bacteria and eukaryotes. Here we present the solution NMR structure of the 102-residue Alr2454 protein from Nostoc sp. PCC 7120, which constitutes the first structural representative from this conserved protein domain family. The structure of Nostoc sp. Alr2454 adopts a novel protein fold.

Solution NMR structures reveal unique homodimer formation by a winged helix-turn-helix motif and provide first structures for protein domain family PF10771.

High-quality NMR structures of the homo-dimeric proteins Bvu3908 (69-residues in monomeric unit) from Bacteroides vulgatus and Bt2368 (74-residues) from Bacteroides thetaiotaomicron reveal the presence of winged helix-turn-helix (wHTH) motifs mediating tight complex formation. Such homo-dimer formation by winged HTH motifs is otherwise found only in two DNA-binding proteins with known structure: the C-terminal wHTH domain of transcriptional activator FadR from E. coli and protein TubR from B. thurigensis, which is involved in plasmid DNA segregation. However, the relative orientation of the wHTH motifs is different and residues involved in DNA-binding are not conserved in Bvu3908 and Bt2368. Hence, the proteins of the present study are not very likely to bind DNA, but are likely to exhibit a function that has thus far not been ascribed to homo-dimers formed by winged HTH motifs. The structures of Bvu3908 and Bt2368 are the first atomic resolution structures for PFAM family PF10771, a family of unknown function (DUF2582) currently containing 128 members.