Computer-aided NMR assay for detecting natively folded structural domains

Structural genomics projects require strategies for rapidly recognizing protein sequences appropriate for routine structure determination. For large proteins, this strategy includes the dissection of proteins into structural domains that form stable native structures. However, protein dissection essentially remains an empirical and often a tedious process. Here, we describe a simple strategy for rapidly identifying structural domains and assessing their structures. This approach combines the computational prediction of sequence regions corresponding to putative domains with an experimental assessment of their structures and stabilities by NMR and biochemical methods. We tested this approach with nine putative domains predicted from a set of 108 Thermus thermophilus HB8 sequences using PASS, a domain prediction program we previously reported. To facilitate the experimental assessment of the domain structures, we developed a generic 6-hour His-tag-based purification protocol, which enables the sample quality evaluation of a putative structural domain in a single day. As a result, we observed that half of the predicted structural domains were indeed natively folded, as judged by their HSQC spectra. Furthermore, two of the natively folded domains were novel, without related sequences classified in the Pfam and SMART databases, which is a significant result with regard to the ability of structural genomics projects to uniformly cover the protein fold space.     

β-Strand-mediated interactions of protein domains

Protein domains exist by themselves or in combination with other domains to form complex multidomain proteins. Defining domain boundaries in proteins is essential for understanding their evolution and function but is not trivial. More specifically, partitioning domains that interact by forming a single β-sheet is known to be particularly troublesome for automatic structure-based domain decomposition pipelines. Here, we study edge-to-edge β-strand interactions between domains in a protein chain, to help define the boundaries for some more difficult cases where a single β-sheet spanning over two domains gives an appearance of one. We give a number of examples where β-strands belonging to a single β-sheet do not belong to a single domain and highlight the difficulties of automatic domain parsers on these examples. This work can be used as a baseline for defining domain boundaries in homologous proteins or proteins with similar domain interactions in the future.     

Conservation of orientation and sequence in protein domain--domain interactions

The repertoire of naturally occurring protein structures is usually characterised in structural terms at the domain level by their constituent folds. As structure is acknowledged to be an important stepping stone to the understanding of protein function, an appreciation of how individual domain interactions are built to form complete, functional protein structures is essential. A comprehensive study of protein domain interactions has been undertaken, covering all those observed in known structures, as well as those predicted to occur in 46 completed genome sequences from all three domains of life. In particular, we examine the promiscuity of protein domains characterised by SCOP superfamilies in terms of their interacting partners, the surface they use to form these interactions, and the relative orientations of their domain partners. Protein domains are shown to display a variety of behaviours, ranging from high promiscuity to absolute monogamy of domain surface employed, with both multiple and single domain partners. In addition, the conservation of sequence and volume at domain interface surfaces is observed to be significantly higher than at accessible surface in general, acting as a powerful potential predictor for domain interactions. We also examine the separation of interacting domains in protein sequence, showing that standard thresholds of 30 amino acid residues lead to a significant false positive rate, and an even more significant false negative rate of approximately 40%. These data suggest that there may be many more than the 2000 domain--domain interactions that have not yet been observed structurally, and we provide a top 30 hit-list of putative domain interactions which should be targeted.     

The structural analysis of protein–protein interactions by NMR spectroscopy

A comprehensive understanding of protein–protein interactions is an important next step in our quest to understand how the information contained in a genome is put into action. Although a number of experimental techniques can report on the existence of a protein– protein interaction, very few can provide detailed structural information. NMR spectroscopy is one of these, and in recent years several complementary NMR approaches, including residual dipolar couplings and the use of paramagnetic effects, have been developed that can provide insight into the structure of protein–protein complexes. In this article, we review these approaches and comment on their strengths and weaknesses.     

Evidence for intermolecular domain exchange in the Fab domains of dimer and oligomers of an IgG1 monoclonal antibody

Recombinant protein therapeutics have become increasingly useful in combating human diseases, such as cancer and those of genetic origin. One quality concern for protein therapeutics is the content and the structure of the aggregated proteins in the product, due to the potential immunogenicity of these aggregates. Collective efforts have led to a better understanding of some types of protein aggregates, and have revealed the diversity in the structure and cause of protein aggregation. In this work we used a broad range of analytical techniques to characterize the quinary structure (complexes in which each composing unit maintains native quaternary structure) of the stable non-covalent dimer and oligomers of a monoclonal IgG1λ antibody. The results supported a mechanism of intermolecular domain exchange involving the Fab domains of 2 or more IgG molecules. This mechanism can account for the native-like higher order (secondary, tertiary and disulfide bonding) structure, the stability of the non-covalent multimers, and the previously observed partial loss of the antigen-binding sites without changing the antigen-binding affinity and kinetics of the remaining sites (Luo et al., 2009, mAbs 1:491). Furthermore, the previously observed increase in the apparent affinity to various Fcγ receptors (ibid), which may potentially promote immunogenicity, was also explained by the quinary structure proposed here. Several lines of evidence indicated that the formation of multimers by the mechanism of intermolecular domain exchange took place mostly during expression, not in the purified materials. The findings in this work will advance our knowledge of the mechanisms for aggregation in therapeutic monoclonal antibodies.     

Comparison of sequence and structure alignments for protein domains

Profile search methods based on protein domain alignments have proven to be useful tools in comparative sequence analysis. Domain alignments used by currently available search methods have been computed by sequence comparison. With the growth of the protein structure database, however, alignments of many domain pairs have also been computed by structure comparison. Here, we examine the extent to which information from these two sources agrees. We measure agreement with respect to identification of homologous regions in each protein, that is, with respect to the location of domain boundaries. We also measure agreement with respect to identification of homologous residue sites by comparing alignments and assessing the accuracy of the molecular models they predict. We find that domain alignments in publicly available collections based on sequence and structure comparison are largely consistent. However, the homologous regions identified by sequence comparison are often shorter than those identified by 3D structure comparison. In addition, when overall sequence similarity is low alignments from sequence comparison produce less accurate molecular models, suggesting that they less accurately identify homologous sites. These observations suggest that structure comparison results might be used to improve the overall accuracy of domain alignment collections and the performance of profile search methods based on them.     

Demonstration of protein-protein interaction specificity by NMR chemical shift mapping.

Chemical shift mapping is becoming a popular method for studying protein-protein interactions in solution. The technique is used to identify putative sites of interaction on a protein surface by detecting chemical shift perturbations in simple (1H, 15N)-HSQC NMR spectra of a uniformly labeled protein as a function of added (unlabeled) target protein. The high concentrations required for these experiments raise questions concerning the possibility for non-specific interactions being detected, thereby compromising the information obtained. We demonstrate here that the simple chemical shift mapping approach faithfully reproduces the known functional specificities among pairs of closely related proteins from the phosphoenolpyruvate:sugar phosphotransferase systems of Escherichia coli and Bacillus subtilis.     

Residual interactions in unfolded bile acid-binding protein by 19F NMR

The folding initiation mechanism of human bile acid-binding protein (BABP) has been examined by (19) F NMR. Equilibrium unfolding studies of BABP labeled with fluorine at all eight of its phenylalanine residues showed that at least two sites experience changes in solvent exposure at high denaturant concentrations. Peak assignments were made by site-specific 4FPhe incorporation. The resonances for proteins specifically labeled at Phe17, Phe47, and Phe63 showed changes in chemical shift at denaturant concentrations at which the remaining five phenylalanine residues appear to be fully solvent-exposed. Phe17 is a helical residue that was not expected to participate in a folding initiation site. Phe47 and Phe63 form part of a hydrophobic core region that may be conserved as a site for folding initiation in the intracellular lipid-binding protein family.     

A novel approach for assessing macromolecular complexes combining soft-docking calculations with NMR data

We present a novel and efficient approach for assessing protein-protein complex formation, which combines ab initio docking calculations performed with the protein docking algorithm BiGGER and chemical shift perturbation data collected with heteronuclear single quantum coherence (HSQC) or TROSY nuclear magnetic resonance (NMR) spectroscopy. This method, termed "restrained soft-docking," is validated for several known protein complexes. These data demonstrate that restrained soft-docking extends the size limitations of NMR spectroscopy and provides an alternative method for investigating macromolecular protein complexes that requires less experimental time, effort, and resources. The potential utility of this novel NMR and simulated docking approach in current structural genomic initiatives is discussed.     

NMR reveals novel mechanisms of protein activity regulation

NMR spectroscopy is one of the most powerful tools for the characterization of biomolecular systems. A unique aspect of NMR is its capacity to provide an integrated insight into both the structure and intrinsic dynamics of biomolecules. In addition, NMR can provide site-resolved information about the conformation entropy of binding, as well as about energetically excited conformational states. Recent advances have enabled the application of NMR for the characterization of supramolecular systems. A summary of mechanisms underpinning protein activity regulation revealed by the application of NMR spectroscopy in a number of biological systems studied in the lab is provided.     

Design and NMR conformational study of a beta-sheet peptide based on Betanova and WW domains

A good approach to test our current knowledge on formation of protein beta-sheets is de novo protein design. To obtain a three-stranded beta-sheet mini-protein, we have built a series of chimeric peptides by taking as a template a previously designed beta-sheet peptide, Betanova-LLM, and incorporating N- and/or C-terminal extensions taken from WW domains, the smallest natural beta-sheet domain that is stable in absence of disulfide bridges. Some Betanova-LLM strand residues were also substituted by those of a prototype WW domain. The designed peptides were cloned and expressed in Escherichia coli. The ability of the purified peptides to adopt beta-sheet structures was examined by circular dichroism (CD). Then, the peptide showing the highest beta-sheet population according to the CD spectra, named 3SBWW-2, was further investigated by 1H and 13C NMR. Based on NOE and chemical shift data, peptide 3SBWW-2 adopts a well defined three-stranded antiparallel beta-sheet structure with a disordered C-terminal tail. To discern between the contributions to beta-sheet stability of strand residues and the C-terminal extension, the structural behavior of a control peptide with the same strand residues as 3SBWW-2 but lacking the C-terminal extension, named Betanova-LYYL, was also investigated. beta-Sheet stability in these two peptides, in the parent Betanova-LLM and in WW-P, a prototype WW domain, decreased in the order WW-P > 3SBWW-2 > Betanova-LYYL > Betanova-LLM. Conclusions about the contributions to beta-sheet stability were drawn by comparing structural properties of these four peptides.     

NMR structure of Citrobacter freundii AmpD,comparison with bacteriophage T7 lysozyme and homology with PGRP domains

AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria. Inactivation of AmpD leads to derepression of beta-lactamase expression, presenting a major pathway for the acquisition of constitutive antibiotic resistance. Here, we report the NMR structure of AmpD from Citrobacter freundii (PDB accession code 1J3G). A deep substrate-binding pocket explains the observed specificity for low molecular mass substrates. The fold is related to that of bacteriophage T7 lysozyme. Both proteins bind zinc at a conserved site and require zinc for amidase activity, although the enzymatic mechanism seems to differ in detail. The structure-based sequence alignment identifies conserved features that are also conserved in the eukaryotic peptidoglycan recognition protein (PGRP) domains, including the zinc-coordination site in several of them. PGRP domains thus belong to the same fold family and, where zinc-binding residues are conserved, may have amidase activity. This hypothesis is supported by the observation that human serum N-acetylmuramyl-L-alanine amidase seems to be identical with a soluble form of human PGRP-L.     

GENFOLD: a genetic algorithm for folding protein structures using NMR restraints.

We report the development and validation of the program GENFOLD, a genetic algorithm that calculates protein structures using restraints obtained from NMR, such as distances derived from nuclear Overhauser effects, and dihedral angles derived from coupling constants. The program has been tested on three proteins: the POU domain (a small three-helix DNA-binding protein), bovine pancreatic trypsin inhibitor (BPTI), and the starch-binding domain from Aspergillus niger glucoamylase I, a 108-residue beta-sheet protein. Structures were calculated for each protein using published NMR restraints. In addition, structures were calculated for BPTI using artificial restraints generated from a high-resolution crystal structure. In all cases the fittest calculated structures were close to the target structure, and could be refined to structures indistinguishable from the target structures by means of a low-temperature simulated annealing refinement. The effectiveness of the program is similar to that of distance geometry and simulated annealing methods, and it is capable of using a very wide range of restraints as input. It can thus be readily extended to the calculation of structures of large proteins, for which few NOE restraints may be available.     

The accuracy of protein structures in solution determined by AlphaFold and NMR

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Finding biologically relevant protein domain interactions: conserved binding mode analysis

Proteins evolved through the shuffling of functional domains, and therefore, the same domain can be found in different proteins and species. Interactions between such conserved domains often involve specific, well-determined binding surfaces reflecting their important biological role in a cell. To find biologically relevant interactions we developed a method of systematically comparing and classifying protein domain interactions from the structural data. As a result, a set of conserved binding modes (CBMs) was created using the atomic detail of structure alignment data and the protein domain classification of the Conserved Domain Database. A conserved binding mode is inferred when different members of interacting domain families dock in the same way, such that their structural complexes superimpose well. Such domain interactions with recurring structural themes have greater significance to be biologically relevant, unlike spurious crystal packing interactions. Consequently, this study gives lower and upper bounds on the number of different types of interacting domain pairs in the structure database on the order of 1000-2000. We use CBMs to create domain interaction networks, which highlight functionally significant connections by avoiding many infrequent links between highly connected nodes. The CBMs also constitute a library of docking templates that may be used in molecular modeling to infer the characteristics of an unknown binding surface, just as conserved domains may be used to infer the structure of an unknown protein. The method's ability to sort through and classify large numbers of putative interacting domain pairs is demonstrated on the oligomeric interactions of globins.     

Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase

The assembly‐line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6‐deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein–protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF₃‐S‐ACP, was developed as a ¹⁹F NMR spectroscopic probe of protein–protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.     

A procedure for detecting structural domains in proteins.

A procedure is described for detecting domains in proteins of known structure. The method is based on the intuitively simple idea that each domain should contain an identifiable hydrophobic core. By applying the algorithm described in the companion paper (Swindells MB, 1995, Protein Sci 4:93-102) to identify distinct cores in multi-domain proteins, one can use this information to determine both the number and the location of the constituent domains. Tests have shown the procedure to be effective on a number of examples, even when the domains are discontinuous along the sequence. However, deficiencies also occur when hydrophobic cores from different domains continue through the interface region and join one another.     

NMR structure of the human doppel protein

The NMR structure of the recombinant human doppel protein, hDpl(24-152), contains a flexibly disordered "tail" comprising residues 24-51, and a globular domain extending from residues 52 to 149 for which a detailed structure was obtained. The globular domain contains four alpha-helices comprising residues 72-80 (alpha1), 101-115 (alpha2(a)), 117-121 (alpha2(b)), and 127-141 (alpha3), and a short two-stranded anti-parallel beta-sheet comprising residues 58-60 (beta1) and 88-90 (beta2). The fold of the hDpl globular domain thus coincides nearly identically with the structure of the murine Dpl protein. There are close similarities with the human prion protein (hPrP) but, similar to the situation with the corresponding murine proteins, hDpl shows marked local differences when compared to hPrP: the beta-sheet is flipped by 180 degrees with respect to the molecular scaffold formed by the four helices, and the beta1-strand is shifted by two residues toward the C terminus. A large solvent-accessible hydrophobic cleft is formed on the protein surface between beta2 and alpha3, which has no counterpart in hPrP. The helix alpha2 of hPrP is replaced by two shorter helices, alpha2(a) and alpha2(b). The helix alpha3 is shortened by more than two turns when compared with alpha3 of hPrP, which is enforced by the positioning of the second disulfide bond in hDpl. The C-terminal peptide segment 144-149 folds back onto the loop connecting beta2 and alpha2. All but four of the 20 conserved residues in the globular domains of hPrP and hDpl appear to have a structural role in maintaining a PrP-type fold. The conservation of R76, E96, N110 and R134 in hDpl, corresponding to R148, E168, N183 and R208 in hPrP suggests that these amino acid residues might have essential roles in the so far unknown functions of PrP and Dpl in healthy organisms.     

Quantitation of protein expression in a cell-free system: Efficient detection of yields and <Superscript>19</Superscript>F NMR to identify folded protein

We have developed an efficient and novel filter assay method, involving radioactive labelling and imaging, to quantify the expression of soluble proteins from a cell-free translation system. Here this method is combined with the conformational sensitivity of ¹⁹F NMR to monitor the folded state of the expressed protein. This report describes the optimisation of 6-fluorotryptophan incorporation in a His-tagged human serum retinol-binding protein (RBP), a disulphide bonded -barrel protein. Appropriate reagent concentrations for producing fluorine labelled RBP in a cell-free translation system are described. It is shown that ¹⁹F NMR is a suitable method for monitoring the production of correctly folded protein from a high-throughput expression system.     

Probing the structure of complex macromolecular interactions by homolog specificity scanning: the P1 and P7 plasmid partition systems.

The P1 plasmid partition locus, P1 par, actively distributes plasmid copies to Escherichia coli daughter cells. It encodes two DNA sites and two proteins, ParA and ParB. Plasmid P7 uses a similar system, but the key macromolecular interactions are species specific. Homolog specificity scanning (HSS) exploits such specificities to map critical contact points between component macromolecules. The ParA protein contacts the par operon operator for operon autoregulation, and the ParB contacts the parS partition site during partition. Here, we refine the mapping of these contacts and extend the use of HSS to map protein-protein contacts. We found that ParB participates in autoregulation at the operator site by making a specific contact with ParA. Similarly, ParA acts in partition by making a specific contact with ParB bound at parS. Both these interactions involve contacts between a C-terminal region of ParA and the extreme N-terminus of ParB. As a single type of ParA-ParB complex appears to be involved in recognizing both DNA sites, the operator and the parS sites may both be occupied by a single protein complex during partition. The general HSS strategy may aid in solving the three-dimensional structures of large complexes of macromolecules.