In this chapter, we concentrate on the production of high-quality protein samples for nuclear magnetic resonance (NMR) studies. In particular, we provide an in-depth description of recent advances in the production of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein production platform of the Northeast Structural Genomics Consortium and outline our high-throughput strategies for producing high-quality protein samples for NMR studies. Our strategy is based on the cloning, expression, and purification of 6×-His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and analytical expression systems, parallel preparative scale fermentation, and high-throughput purification protocols. The 6×-His affinity tag allows for a similar two-step purification procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (>97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5000 of these proteins have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html), resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this chapter describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are suitable for implementation in a large individual laboratory or by a small group of collaborating investigators for structural biology, functional proteomics, ligand screening, and structural genomics research.

Bacterial toxin-antitoxin (TA) systems serve a variety of physiological functions including regulation of cell growth and maintenance of foreign genetic elements. Sequence analyses suggest that TA families are linked by complex evolutionary relationships reflecting likely swapping of functional domains between different TA families. Our crystal structures of Phd-Doc from bacteriophage P1, the HigA antitoxin from Escherichia coli CFT073, and YeeU of the YeeUWV systems from E. coli K12 and Shigella flexneri confirm this inference and reveal additional, unanticipated structural relationships. The growth-regulating Doc toxin exhibits structural similarity to secreted virulence factors that are toxic for eukaryotic target cells. The Phd antitoxin possesses the same fold as both the YefM and NE2111 antitoxins that inhibit structurally unrelated toxins. YeeU, which has an antitoxin-like activity that represses toxin expression, is structurally similar to the ribosome-interacting toxins YoeB and RelE. These observations suggest extensive functional exchanges have occurred between TA systems during bacterial evolution.

Target-site selection by retroviral integrase (IN) proteins profoundly affects viral pathogenesis. We describe the solution nuclear magnetic resonance structure of the Moloney murine leukemia virus IN (M-MLV) C-terminal domain (CTD) and a structural homology model of the catalytic core domain (CCD). In solution, the isolated MLV IN CTD adopts an SH3 domain fold flanked by a C-terminal unstructured tail. We generated a concordant MLV IN CCD structural model using SWISS-MODEL, MMM-tree and I-TASSER. Using the X-ray crystal structure of the prototype foamy virus IN target capture complex together with our MLV domain structures, residues within the CCD α2 helical region and the CTD β1-β2 loop were predicted to bind target DNA. The role of these residues was analyzed in vivo through point mutants and motif interchanges. Viable viruses with substitutions at the IN CCD α2 helical region and the CTD β1-β2 loop were tested for effects on integration target site selection. Next-generation sequencing and analysis of integration target sequences indicate that the CCD α2 helical region, in particular P187, interacts with the sequences distal to the scissile bonds whereas the CTD β1-β2 loop binds to residues proximal to it. These findings validate our structural model and disclose IN-DNA interactions relevant to target site selection.

Repeat proteins have considerable potential for use as modular binding reagents or biomaterials in biomedical and nanotechnology applications. Here we describe a general computational method for building idealized repeats that integrates available family sequences and structural information with Rosetta de novo protein design calculations. Idealized designs from six different repeat families were generated and experimentally characterized; 80% of the proteins were expressed and soluble and more than 40% were folded and monomeric with high thermal stability. Crystal structures determined for members of three families are within 1Å root-mean-square deviation to the design models. The method provides a general approach for fast and reliable generation of stable modular repeat protein scaffolds.

We have found that refinement of protein NMR structures using Rosetta with experimental NMR restraints yields more accurate protein NMR structures than those that have been deposited in the PDB using standard refinement protocols. Using 40 pairs of NMR and X-ray crystal structures determined by the Northeast Structural Genomics Consortium, for proteins ranging in size from 5-22 kDa, restrained Rosetta refined structures fit better to the raw experimental data, are in better agreement with their X-ray counterparts, and have better phasing power compared to conventionally determined NMR structures. For 37 proteins for which NMR ensembles were available and which had similar structures in solution and in the crystal, all of the restrained Rosetta refined NMR structures were sufficiently accurate to be used for solving the corresponding X-ray crystal structures by molecular replacement. The protocol for restrained refinement of protein NMR structures was also compared with restrained CS-Rosetta calculations. For proteins smaller than 10 kDa, restrained CS-Rosetta, starting from extended conformations, provides slightly more accurate structures, while for proteins in the size range of 10-25 kDa the less CPU intensive restrained Rosetta refinement protocols provided equally or more accurate structures. The restrained Rosetta protocols described here can improve the accuracy of protein NMR structures and should find broad and general for studies of protein structure and function.

We have determined the solution NMR structure of the intermembrane space domain (IMSD) of the human mitochondrial ATPase associated with various activities (AAA) protease known as AFG3-like protein 2 (AFG3L2). Our structural analysis and molecular dynamics results indicate that the IMSD is peripherally bound to the membrane surface. This is a modification to the location of the six IMSDs in a model of the full length yeast hexaoligomeric homolog of AFG3L2 determined at low resolution by electron cryomicroscopy [1]. The predicted protein-protein interaction surface, located on the side furthest from the membrane, may mediate binding to substrates as well as prohibitins.

The genomes of more than 50 organisms have now been manipulated due to rapid advancement of gene editing technology. One way to perform gene editing in the mouse using the CRISPR/CAS system, guide RNA (gRNA) and CAS9 mRNA transcribed in vitro are microinjected into fertilized eggs that are then allowed to develop to term. As a rule, gRNAs are tested first in tissue culture cells and the one with the highest locus-specific cleavage activity is chosen for microinjection. For cell transfections, gRNAs are typically expressed using the human U6 promoter (hU6). However, gRNAs for microinjection into zygotes are obtained by in vitro transcription from a T7 bacteriophage promoter in a separate plasmid vector. Here, we describe the design and construction of a combined U6T7 hybrid promoter from which the same gRNA sequence can be expressed. An expression vector containing such a hybrid promoter can now be used to generate gRNA for testing in mammalian cells as well as for microinjection purposes. The gRNAs expressed and transcribed from this vector are found to be functional in cells as well as in mice.

The biochemical and physical factors controlling protein expression level and solubility in vivo remain incompletely characterized. To gain insight into the primary sequence features influencing these outcomes, we performed statistical analyses of results from the high-throughput protein-production pipeline of the Northeast Structural Genomics Consortium. Proteins expressed in E. coli and consistently purified were scored independently for expression and solubility levels. These parameters nonetheless show a very strong positive correlation. We used logistic regressions to determine whether they are systematically influenced by fractional amino acid composition or several bulk sequence parameters including hydrophobicity, sidechain entropy, electrostatic charge, and predicted backbone disorder. Decreasing hydrophobicity correlates with higher expression and solubility levels, but this correlation apparently derives solely from the beneficial effect of three charged amino acids, at least for bacterial proteins. In fact, the three most hydrophobic residues showed very different correlations with solubility level. Leu showed the strongest negative correlation among amino acids, while Ile showed a slightly positive correlation in most data segments. Several other amino acids also had unexpected effects. Notably, Arg correlated with decreased expression and, most surprisingly, solubility of bacterial proteins, an effect only partially attributable to rare codons. However, rare codons did significantly reduce expression despite use of a codon-enhanced strain. Additional analyses suggest that positively but not negatively charged amino acids may reduce translation efficiency in E. coli irrespective of codon usage. While some observed effects may reflect indirect evolutionary correlations, others may reflect basic physicochemical phenomena. We used these results to construct and validate predictors of expression and solubility levels and overall protein usability, and we propose new strategies to be explored for engineering improved protein expression and solubility.

No abstract available

Cell surface molecules are important for development and function of multicellular organisms. Although several methods are available to identify ligand-receptor pairs, ELISA-based methods are particularly amenable to high-throughput screens. ELISA-based methods have high sensitivity and low false-positive rates for detecting protein-protein interaction (PPI) complexes. Here, we provide a detailed protocol for a 384-well ELISA-based PPI screening protocol for the identification of novel cell surface ligand-receptor interactions, together with considerations for validation of PPIs by biophysical methods. This PPI screen has been developed and tested for discovery of novel ligand-receptor pairs between human synaptic adhesion proteins, believed to play crucial roles in many steps of neurodevelopment, from neuronal maturation, to axon guidance, synapse connectivity, and pruning.

Online citizen science projects such as GalaxyZoo1, Eyewire2 and Phylo3 have proven very successful for data collection, annotation and processing, but for the most part have harnessed human pattern-recognition skills rather than human creativity. An exception is the game EteRNA4, in which game players learn to build new RNA structures by exploring the discrete two-dimensional space of Watson-Crick base pairing possibilities. Building new proteins, however, is a more challenging task to present in a game, as both the representation and evaluation of a protein structure are intrinsically three-dimensional. We posed the challenge of de novo protein design in the online protein-folding game Foldit5. Players were presented with a fully extended peptide chain and challenged to craft a folded protein structure and an amino acid sequence encoding that structure. After many iterations of player design, analysis of the top-scoring solutions and subsequent game improvement, Foldit players can now-starting from an extended polypeptide chain-generate a diversity of protein structures and sequences that encode them in silico. One hundred forty-six Foldit player designs with sequences unrelated to naturally occurring proteins were encoded in synthetic genes; 56 were found to be expressed and soluble in Escherichia coli, and to adopt stable monomeric folded structures in solution. The diversity of these structures is unprecedented in de novo protein design, representing 20 different folds-including a new fold not observed in natural proteins. High-resolution structures were determined for four of the designs, and are nearly identical to the player models. This work makes explicit the considerable implicit knowledge that contributes to success in de novo protein design, and shows that citizen scientists can discover creative new solutions to outstanding scientific challenges such as the protein design problem.

The extraterminal (ET) domain of BRD3 is conserved among BET proteins (BRD2, BRD3, BRD4), interacting with multiple host and viral protein-protein networks. Solution NMR structures of complexes formed between the BRD3 ET domain and either the 79-residue murine leukemia virus integrase (IN) C-terminal domain (IN329-408) or its 22-residue IN tail peptide (IN386-407) alone reveal similar intermolecular three-stranded β-sheet formations. 15N relaxation studies reveal a 10-residue linker region (IN379-388) tethering the SH3 domain (IN329-378) to the ET-binding motif (IN389-405):ET complex. This linker has restricted flexibility, affecting its potential range of orientations in the IN:nucleosome complex. The complex of the ET-binding peptide of the host NSD3 protein (NSD3148-184) and the BRD3 ET domain includes a similar three-stranded β-sheet interaction, but the orientation of the β hairpin is flipped compared with the two IN:ET complexes. These studies expand our understanding of molecular recognition polymorphism in complexes of ET-binding motifs with viral and host proteins.

Recent advances in molecular modeling of protein structures are changing the field of structural biology. AlphaFold-2 (AF2), an AI system developed by DeepMind, Inc., utilizes attention-based deep learning to predict models of protein structures with high accuracy relative to structures determined by X-ray crystallography and cryo-electron microscopy (cryoEM). Comparing AF2 models to structures determined using solution NMR data, both high similarities and distinct differences have been observed. Since AF2 was trained on X-ray crystal and cryoEM structures, we assessed how accurately AF2 can model small, monomeric, solution protein NMR structures which (i) were not used in the AF2 training data set, and (ii) did not have homologous structures in the Protein Data Bank at the time of AF2 training. We identified nine open source protein NMR data sets for such "blind" targets, including chemical shift, raw NMR FID data, NOESY peak lists, and (for 1 case) 15 N- 1 H residual dipolar coupling data. For these nine small (70 - 108 residues) monomeric proteins, we generated AF2 prediction models and assessed how well these models fit to these experimental NMR data, using several well-established NMR structure validation tools. In most of these cases, the AF2 models fit the NMR data nearly as well, or sometimes better than, the corresponding NMR structure models previously deposited in the Protein Data Bank. These results provide benchmark NMR data for assessing new NMR data analysis and protein structure prediction methods. They also document the potential for using AF2 as a guiding tool in protein NMR data analysis, and more generally for hypothesis generation in structural biology research.

Identifying the interactome for a protein of interest is challenging due to the large number of possible binders. High-throughput experimental approaches narrow down possible binding partners, but often include false positives. Furthermore, they provide no information about what the binding region is (e.g. the binding epitope). We introduce a novel computational pipeline based on an AlphaFold2 (AF) Competition Assay (AF-CBA) to identify proteins that bind a target of interest from a pull-down experiment, along with the binding epitope. Our focus is on proteins that bind the Extraterminal (ET) domain of Bromo and Extraterminal domain (BET) proteins, but we also introduce nine additional systems to show transferability to other peptide-protein systems. We describe a series of limitations to the methodology based on intrinsic deficiencies to AF and AF-CBA, to help users identify scenarios where the approach will be most useful. Given the speed and accuracy of the methodology, we expect it to be generally applicable to facilitate target selection for experimental verification starting from high-throughput protein libraries.

The DNA-damage repair pathway homologous recombination (HR) requires factors that promote the activity of strand-exchange protein RAD51 and its meiosis-specific homolog DMC1. Here we show that the Shu complex SWS1-SWSAP1, a candidate for one such HR regulator, is dispensable for mouse viability but essential for male and female fertility, promoting the assembly of RAD51 and DMC1 on early meiotic HR intermediates. Only a fraction of mutant meiocytes progress to form crossovers, which are crucial for chromosome segregation, demonstrating crossover homeostasis. Remarkably, loss of the DNA damage checkpoint kinase CHK2 rescues fertility in females without rescuing crossover numbers. Concomitant loss of the BRCA2 C terminus aggravates the meiotic defects in Swsap1 mutant spermatocytes, suggesting an overlapping role with the Shu complex during meiotic HR. These results demonstrate an essential role for SWS1-SWSAP1 in meiotic progression and emphasize the complex interplay of factors that ensure recombinase function.

We performed a comprehensive structure validation of both automated and manually generated structures of the 10 targets of the CASD-NMR-2013 effort. We established that automated structure determination protocols are capable of reliably producing structures of comparable accuracy and quality to those generated by a skilled researcher, at least for small, single domain proteins such as the ten targets tested. The most robust results appear to be obtained when NOESY peak lists are used either as the primary input data or to augment chemical shift data without the need to manually filter such lists. A detailed analysis of the long-range NOE restraints generated by the different programs from the same data showed a surprisingly low degree of overlap. Additionally, we found that there was no significant correlation between the extent of the NOE restraint overlap and the accuracy of the structure. This result was surprising given the importance of NOE data in producing good quality structures. We suggest that this could be explained by the information redundancy present in NOEs between atoms contained within a fixed covalent network.

RAS binding is a critical step in the activation of BRAF protein serine/threonine kinase and stimulation of the mitogen-activated protein kinase signaling pathway. Mutations in both RAS and BRAF are associated with many human cancers. Here, we report the solution nuclear magnetic resonance (NMR) and X-ray crystal structures of the RAS-binding domain (RBD) from human BRAF. We further studied the complex between BRAF RBD and the GppNHp bound form of HRAS in solution. Backbone, side-chain, and (19)F NMR chemical shift perturbations reveal unexpected changes distal to the RAS-binding face that extend through the core of the RBD structure. Moreover, backbone amide hydrogen/deuterium exchange NMR data demonstrate conformational ensemble changes in the RBD core structure upon complex formation. These changes in BRAF RBD reveal a basis for allosteric regulation of BRAF structure and function, and suggest a mechanism by which RAS binding can signal the drastic domain rearrangements required for activation of BRAF kinase.

Molecular replacement (MR) is widely used for addressing the phase problem in X-ray crystallography. Historically, crystallographers have had limited success using NMR structures as MR search models. Here, we report a comprehensive investigation of the utility of protein NMR ensembles as MR search models, using data for 25 pairs of X-ray and NMR structures solved and refined using modern NMR methods. Starting from NMR ensembles prepared by an improved protocol, FindCore, correct MR solutions were obtained for 22 targets. Based on these solutions, automatic model rebuilding could be done successfully. Rosetta refinement of NMR structures provided MR solutions for another two proteins. We also demonstrate that such properly prepared NMR ensembles and X-ray crystal structures have similar performance when used as MR search models for homologous structures, particularly for targets with sequence identity >40%.

During meiotic prophase in male mammals, the X and Y chromosomes condense to form a macrochromatin body, termed the sex, or XY, body, within which X- and Y-linked genes are transcriptionally repressed. The molecular basis and biological function of both sex body formation and meiotic sex chromosome inactivation (MSCI) are unknown. A phosphorylated form of H2AX, a histone H2A variant implicated in DNA repair, accumulates in the sex body in a manner independent of meiotic recombination-associated double-strand breaks. Here we show that the X and Y chromosomes of histone H2AX-deficient spermatocytes fail to condense to form a sex body, do not initiate MSCI, and exhibit severe defects in meiotic pairing. Moreover, other sex body proteins, including macroH2A1.2 and XMR, do not preferentially localize with the sex chromosomes in the absence of H2AX. Thus, H2AX is required for the chromatin remodeling and associated silencing in male meiosis.

Recent developments provide automated analysis of NMR assignments and three-dimensional (3D) structures of proteins. These approaches are generally applicable to proteins ranging from about 50 to 150 amino acids. In this chapter, we summarize progress by the Northeast Structural Genomics Consortium in standardizing the NMR data collection process for protein structure determination and in building an integrated platform for automated protein NMR structure analysis. Our integrated platform includes the following principal steps: (1) standardized NMR data collection, (2) standardized data processing (including spectral referencing and Fourier transformation), (3) automated peak picking and peak list editing, (4) automated analysis of resonance assignments, (5) automated analysis of NOESY data together with 3D structure determination, and (6) methods for protein structure validation. In particular, the software AutoStructure for automated NOESY data analysis is described in this chapter, together with a discussion of practical considerations for its use in high-throughput structure production efforts. The critical area of data quality assessment has evolved significantly over the past few years and involves evaluation of both intermediate and final peak lists, resonance assignments, and structural information derived from the NMR data. Methods for quality control of each of the major automated analysis steps in our platform are also discussed. Despite significant remaining challenges, when good quality data are available, automated analysis of protein NMR assignments and structures with this platform is both fast and reliable.