Paolo Rossi

Consistent blind protein structure generation from NMR chemical shift data

Protein NMR chemical shifts are highly sensitive to local structure. A robust protocol is described that exploits this relation for de novo protein structure generation, using as input experimental parameters the 13Cα, 13Cβ, 13C′, 15N, 1Hα and 1HN NMR chemical shifts. These shifts are generally available at the early stage of the traditional NMR structure determination process, before the collection and analysis of structural restraints. The chemical shift based structure determination protocol uses an empirically optimized procedure to select protein fragments from the Protein Data Bank, in conjunction with the standard ROSETTA Monte Carlo assembly and relaxation methods. Evaluation of 16 proteins, varying in size from 56 to 129 residues, yielded full-atom models that have 0.7–1.8 Å root mean square deviations for the backbone atoms relative to the experimentally determined x-ray or NMR structures. The strategy also has been successfully applied in a blind manner to nine protein targets with molecular masses up to 15.4 kDa, whose conventional NMR structure determination was conducted in parallel by the Northeast Structural Genomics Consortium. This protocol potentially provides a new direction for high-throughput NMR structure determination.

NMR structure determination for larger proteins using backbone-only data.

Examining the Backbone Determination of tertiary protein structures by nuclear magnetic resonance (NMR) currently relies heavily on side-chain NMR data. The assignment of side-chain atoms is challenging. In addition, proteins larger than 15 kilodaltons (kD) must be deuterated to improve resolution and this eliminates the possibility of measuring long-range interproton distance constraints. Now Raman et al. (p. 1014, published online 4 February) use backbone-only NMR data—chemical shifts, residual dipolar coupling, and backbone amide proton distances—available from highly deuterated proteins to guide conformational searching in the Rosetta structure prediction protocol. Using this new protocol, they were able to generate accurate structures for proteins of up to 25 kD. Protein structures can be determined by using the limited nuclear magnetic resonance information obtainable for larger proteins. Conventional protein structure determination from nuclear magnetic resonance data relies heavily on side-chain proton-to-proton distances. The necessary side-chain resonance assignment, however, is labor intensive and prone to error. Here we show that structures can be accurately determined without nuclear magnetic resonance (NMR) information on the side chains for proteins up to 25 kilodaltons by incorporating backbone chemical shifts, residual dipolar couplings, and amide proton distances into the Rosetta protein structure modeling methodology. These data, which are too sparse for conventional methods, serve only to guide conformational search toward the lowest-energy conformations in the folding landscape; the details of the computed models are determined by the physical chemistry implicit in the Rosetta all-atom energy function. The new method is not hindered by the deuteration required to suppress nuclear relaxation processes for proteins greater than 15 kilodaltons and should enable routine NMR structure determination for larger proteins.

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.

Structural Basis for Protein Antiaggregation Activity of the Trigger Factor Chaperone

Introduction Molecular chaperones prevent aggregation and misfolding of proteins in the cellular environment and are thus central to maintaining protein homeostasis. Molecular chaperones are thought to recognize and bind to exposed hydrophobic regions of the unfolded proteins, thereby shielding these regions from the solvent. If unprotected, the proteins would likely aggregate or misfold to bury the hydrophobic residues. Despite the central importance of the binding of chaperones to unfolded proteins, the structural basis of their interaction remains poorly understood. The scarcity of structural data on complexes between chaperones and unfolded proteins is primarily due to technical challenges originating in the size and dynamic nature of these complexes. Structural basis of PhoA binding by TF. PhoA (blue/gray) is captured in an unfolded state by three TF chaperone molecules (orange). Complex formation is mediated by multivalent binding of hydrophobic surfaces, which are shielded from water, thereby preventing folding and, at the same time, aggregation of the substrate protein.Structural basis of PhoA binding by TF. PhoA (blue/gray) is captured in an unfolded state by three TF chaperone molecules (orange). Complex formation is mediated by multivalent binding of hydrophobic surfaces, which are shielded from water, thereby preventing folding and, at the same time, aggregation of the substrate protein. Rationale Recent advances in nuclear magnetic resonance (NMR) and isotope labeling approaches make it possible to study large, dynamic complexes. We used NMR spectroscopy to characterize the binding of the 48-kD unfolded alkaline phosphatase (PhoA) to the 50-kD trigger factor (TF) chaperone. We obtained atomic insight into the dynamic binding and determined the solution structure of PhoA captured in an extended, unfolded state by three TF molecules. Based on our NMR studies, we gained insight into how TF rescues an aggregation-prone protein and how it exerts its unfoldase activity. Results We show that TF uses multiple sites, which are located in two different domains and extend over a distance of ~90 Å, to bind to several regions of the unfolded PhoA that are dispersed throughout its entire length. Three TF molecules are required to interact with the entire length of PhoA, giving rise to a ~200-kD complex in solution. The TF-PhoA interactions are mediated primarily by hydrophobic contacts. TF interacts with PhoA in a highly dynamic fashion, giving rise to a rugged landscape for the free energy of interaction. As the number and length of the PhoA regions engaged by TF increases, a more stable complex gradually emerges. The multivalent binding keeps PhoA in an extended, unfolded conformation. Crucially, even the lowest-energy TF-PhoA complex remains rather dynamic with a lifetime of ~20 ms. The structural data of the three TF molecules in complex with different regions of PhoA reveal how the same binding sites within a molecular chaperone can recognize and interact with a large number of substrates with unrelated primary sequences. This promiscuous recognition is further enabled by the notable plasticity of the substrate-binding sites in TF. We finally show that TF in the cytosol prevents aggregation by interacting transiently with the low-populated, aggregation-prone unfolded state of the substrate but acts as a powerful unfoldase when it is bound at the ribosome and thus is colocalized with translating substrate. Conclusion The structural data reveal a multivalent binding mechanism between the chaperone and its protein substrate. This mechanism of binding presents several advantages as it enables chaperones to function as holdases and unfoldases by exerting forces to retain proteins in the unfolded state and at the same time protect them from aggregation by shielding their exposed hydrophobic regions. Given the existence of multiple binding sites in other molecular chaperones, this may present a general mechanism for the action of molecular chaperones. The fast kinetics of substrate binding enables chaperones to interact with transiently exposed, aggregation-prone regions of unstable proteins in the cytosol, thereby preventing their aggregation and increasing their solubility. Nuclear magnetic resonance data show how molecular chaperones recognize and prevent aggregation and misfolding of unfolded proteins. [Also see Perspective by Gamerdinger and Deuerling] Molecular chaperones prevent aggregation and misfolding of proteins, but scarcity of structural data has impeded an understanding of the recognition and antiaggregation mechanisms. We report the solution structure, dynamics, and energetics of three trigger factor (TF) chaperone molecules in complex with alkaline phosphatase (PhoA) captured in the unfolded state. Our data show that TF uses multiple sites to bind to several regions of the PhoA substrate protein primarily through hydrophobic contacts. Nuclear magnetic resonance (NMR) relaxation experiments show that TF interacts with PhoA in a highly dynamic fashion, but as the number and length of the PhoA regions engaged by TF increase, a more stable complex gradually emerges. Multivalent binding keeps the substrate protein in an extended, unfolded conformation. The results show how molecular chaperones recognize unfolded polypeptides and, by acting as unfoldases and holdases, prevent the aggregation and premature (mis)folding of unfolded proteins. Recognize and Protect Molecular chaperones play a key role in maintaining protein homeostasis in the cell by preventing protein aggregation and misfolding. Chaperone-substrate complexes tend to be large and dynamic, making structure determination challenging. Saio et al. (10.1126/science.1250494; see the Perspective by Gamerdinger and Deuerling) used advanced NMR spectroscopy techniques to determine the structure of three trigger factor (TF) chaperone molecules in complex with the unfolded substrate, alkaline phosphatase (PhoA), and of each of the TFs in complex with the relevant region of PhoA. TF binds at multiple sites on PhoA through hydrophobic contacts, thus shielding these residues from solvent and preventing aggregation. The stability of the complex increases as longer PhoA regions are engaged by TF, and the multivalent binding keeps the substrate in an extended conformation.

The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium

We describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples. The platform is centered on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems. The 6X-His tag allows for similar purification procedures for most targets and implementation of high-throughput (HTP) parallel methods. In most cases, the 6X-His-tagged proteins are sufficiently purified (>97% homogeneity) using a HTP two-step purification protocol for most structural studies. Using this platform, the open reading frames of over 16,000 different targeted proteins (or domains) have been cloned as>26,000 constructs. Over the past 10 years, more than 16,000 of these expressed protein, and more than 4400 proteins (or domains) have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html). Using these samples, the NESG has deposited more than 900 new protein structures to the Protein Data Bank (PDB). The methods described here are effective in producing eukaryotic and prokaryotic protein samples in E. coli. This paper summarizes some of the updates made to the protein production pipeline in the last 5 years, corresponding to phase 2 of the NIGMS Protein Structure Initiative (PSI-2) project. The NESG Protein Production Platform is suitable for implementation in a large individual laboratory or by a small group of collaborating investigators. These advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are of broad value to the structural biology, functional proteomics, and structural genomics communities.

Blind Testing of Routine, Fully Automated Determination of Protein Structures from NMR Data

The protocols currently used for protein structure determination by nuclear magnetic resonance (NMR) depend on the determination of a large number of upper distance limits for proton-proton pairs. Typically, this task is performed manually by an experienced researcher rather than automatically by using a specific computer program. To assess whether it is indeed possible to generate in a fully automated manner NMR structures adequate for deposition in the Protein Data Bank, we gathered 10 experimental data sets with unassigned nuclear Overhauser effect spectroscopy (NOESY) peak lists for various proteins of unknown structure, computed structures for each of them using different, fully automatic programs, and compared the results to each other and to the manually solved reference structures that were not available at the time the data were provided. This constitutes a stringent blind assessment similar to the CASP and CAPRI initiatives. This study demonstrates the feasibility of routine, fully automated protein structure determination by NMR.

Preparation of protein samples for NMR structure, function, and small-molecule screening studies.

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.

Solution NMR structure of the NlpC/P60 domain of lipoprotein Spr from Escherichia coli: structural evidence for a novel cysteine peptidase catalytic triad.

Escherichia coli Spr is a membrane-anchored cell wall hydrolase. The solution NMR structure of the C-terminal NlpC/P60 domain of E. coli Spr described here reveals that the protein adopts a papain-like alpha+beta fold and identifies a substrate-binding cleft featuring several highly conserved residues. The active site features a novel Cys-His-His catalytic triad that appears to be a unique structural signature of this cysteine peptidase family. Moreover, the relative orientation of these catalytic residues is similar to that observed in the analogous Ser-His-His triad, a variant of the classic Ser-His-Asp charge relay system, suggesting the convergent evolution of a catalytic mechanism in quite distinct peptidase families.

A microscale protein NMR sample screening pipeline

As part of efforts to develop improved methods for NMR protein sample preparation and structure determination, the Northeast Structural Genomics Consortium (NESG) has implemented an NMR screening pipeline for protein target selection, construct optimization, and buffer optimization, incorporating efficient microscale NMR screening of proteins using a micro-cryoprobe. The process is feasible because the newest generation probe requires only small amounts of protein, typically 30–200xa0μg in 8–35xa0μl volume. Extensive automation has been made possible by the combination of database tools, mechanization of key process steps, and the use of a micro-cryoprobe that gives excellent data while requiring little optimization and manual setup. In this perspective, we describe the overall process used by the NESG for screening NMR samples as part of a sample optimization process, assessing optimal construct design and solution conditions, as well as for determining protein rotational correlation times in order to assess protein oligomerization states. Database infrastructure has been developed to allow for flexible implementation of new screening protocols and harvesting of the resulting output. The NESG micro NMR screening pipeline has also been used for detergent screening of membrane proteins. Descriptions of the individual steps in the NESG NMR sample design, production, and screening pipeline are presented in the format of a standard operating procedure.

Determination of the Structures of Symmetric Protein Oligomers from NMR Chemical Shifts and Residual Dipolar Couplings

Symmetric protein dimers, trimers, and higher-order cyclic oligomers play key roles in many biological processes. However, structural studies of oligomeric systems by solution NMR can be difficult due to slow tumbling of the system and the difficulty in identifying NOE interactions across protein interfaces. Here, we present an automated method (RosettaOligomers) for determining the solution structures of oligomeric systems using only chemical shifts, sparse NOEs, and domain orientation restraints from residual dipolar couplings (RDCs) without a need for a previously determined structure of the monomeric subunit. The method integrates previously developed Rosetta protocols for solving the structures of monomeric proteins using sparse NMR data and for predicting the structures of both nonintertwined and intertwined symmetric oligomers. We illustrated the performance of the method using a benchmark set of nine protein dimers, one trimer, and one tetramer with available experimental data and various interface topologies. The final converged structures are found to be in good agreement with both experimental data and previously published high-resolution structures. The new approach is more readily applicable to large oligomeric systems than conventional structure-determination protocols, which often require a large number of NOEs, and will likely become increasingly relevant as more high-molecular weight systems are studied by NMR.