How the Protein Data Bank Changed Biology: An Introduction to The JBC Reviews Thematic Series, Part 1

This collection of articles celebrates the 50th anniversary of the Protein Data Bank (PDB), the single global digital archive of biological macromolecular structures. The impact of the PDB is immense; we have invited a number of top researchers in structural biology to illustrate its influence on an array of scientific fields. What emerges is a compelling picture of the synergism between the PDB and the explosive progress witnessed in many scientific areas. Availability of reliable, openly accessible, well-archived structural information has arguably had more impact on cell and molecular biology than even some of the enabling technologies such as PCR. We have seen the science move from a time when structural biologists contributed the lion\u27s share of the structures to the PDB and for discussion within their community to a time when any effort to achieve in-depth understanding of a biochemical or cell biological question demands an interdisciplinary approach built atop structural underpinnings

Protein folding in the cell: challenges and progress.

It is hard to imagine a more extreme contrast than that between the dilute solutions used for in vitro studies of protein folding and the crowded, compartmentalized, sticky, spatially inhomogeneous interior of a cell. This review highlights recent research exploring protein folding in the cell with a focus on issues that are generally not relevant to in vitro studies of protein folding, such as macromolecular crowding, hindered diffusion, cotranslational folding, molecular chaperones, and evolutionary pressures. The technical obstacles that must be overcome to characterize protein folding in the cell are driving methodological advances, and we draw attention to several examples, such as fluorescence imaging of folding in cells and genetic screens for in-cell stability. DOI 10.1016DOI 10. /j.sbi.2010 Introduction Chris Anfinsen launched the field of protein folding by showing that ribonuclease (specifically, bovine pancreatic ribonuclease A) could refold to an active enzyme after reductive denaturation. Naturally, ribonuclease became emblematic of the fundamental tenet of protein folding -that the primary sequence of a protein specifies an energy landscape and a successful route to the native state at the global energy minimum. Yet ribonuclease folds in vivo during a complex journey through the secretory pathway of the cell. Notably, in its biological folding process, ribonuclease confronts milieux that are densely crowded with macromolecules; it samples the microenvironments of the ribosome tunnel, the translocon, and the ER lumen; it has the opportunity to fold from its N-terminus to C-terminus; and it is not left on its own, but instead is accompanied by lumenal chaperones that facilitate its folding and post-translational modification. As this journey abundantly illustrates, protein folding in the cell confronts many issues that are nonexistent in high dilution refolding experiments. It is thus not surprising that an increasing research effort is being applied to issues and processes involved in cellular protein folding. This review presents research in the expanding area of protein folding in the cell. We mainly confine our discussion to publications on cellular protein folding that have appeared in the last two years and to areas that have not been recently reviewed. We first describe a number of issues, such as macromolecular crowding and cotranslational folding, that arise when considering folding in the cell but are absent in vitro ( Macromolecular crowding A striking difference between most in vitro folding experiments and the cellular environment is the high concentration of macromolecules, which severely limits the cellular volume accessible to a polypeptide chain. Like many issues related to folding in the cell, determining the effects of crowding on folding presents major technical challenges to both computational and experimental studies; moreover, crowding is generally accompanied by other effects including altered diffusion and weak interactions. Macromolecular crowding also affects the viscosity of the cellular environment and solvent viscosity has been invoked as an important factor in determining folding rates and mechanisms (e.g., [9]). In a recent study, Dhar et al. used computational and experimental approaches to study the effects of a model crowder, Ficoll, on activity and folding of phosphoglycerate kinase (PGK), a 412-aa protein with two domains connected by a flexible hinge www.sciencedirect.com Current Opinion in Structural Biology 2011, 21:32-41 decreased the inter-domain separation. Also, the relaxation rate from temperature-jump unfolding experiments showed a maximum at 100 g/L Ficoll. The authors interpreted this result as arising from opposing effects of crowding and viscosity Hindered mobility and sticky neighbors Several recent studies show that the cellular environment affects macromolecular motion and provide insight into how. For example, two recent papers have used fluorescence recovery after photobleaching to monitor translational diffusion of GFP constructs in E. coli cells Vectorial synthesis and roles of mRNA and ribosomes in folding Newly synthesized polypeptide chains emerge from the ribosome vectorially, allowing their N-terminal portions to sample conformational space before the chain is completely synthesized. Additionally, the earliest environments encountered by a nascent chain are the ribosome tunnel and ribosome-associated chaperones. There have been excellent recent reviews on issues related to cotranslational folding including one in this issue A single domain stabilized by many long-range contacts is not expected to fold until the entire chain is complete, and recent studies of ribosome-bound nascent chains (RNCs) have confirmed this expectation for an SH3 domain by NMR [20] and GFP by observing chromophore maturation [21 ]. In fact, the NMR studies on SH3 RNCs reveal little or no compaction until the entire chain has exited the ribosomal tunnel [20]. By comparison, RNCs of the larger GFP may populate a more compact state before full translation [21 ]. The interrelationship of translation rate and folding has been discussed in a number of recent reviews [22][23][24][25]. Intuitively, slowing translation might allow more time for proper folding, and indeed in a recent study mutant ribosomes with reduced translation rates increased the soluble expression of eukaryotic proteins in E. coli [26 ]. The messenger RNA sequence can also affect the translation rate either through the use of rare codons [22,25,[27][28][29] or by RNA folding [29,30]. Either of these factors may be changed by synonymous mutations, where mRNA sequences are altered without affecting the encoded amino acid. Increasing the translation rate of the multidomain E. coli protein SufI by synonymously exchanging rare codon clusters for common codons was found to decrease cotranslational folding and the production of mature, folded protein [31 ]. Intriguingly, the mutation that leads to the deletion of F508 (DF508) in the cystic fibrosis transmembrane conductance regulator (CFTR), the most common mutation linked to cystic fibrosis, also changes the preceding codon for Ile507 [32 ]. Alteration of the local mRNA structure in the mutant retards translation and presumably impairs folding, increasing cotranslational ubiquitination and leading to protein degradation [32 ,33]. Restoration of the original Ile507 codon in the DF508 background significantly increases the amount of mature CFTR in the plasma membrane, demonstrating the potential impact of synonymous mutations on in vivo protein folding and maturation [32 ,33]. As described in recent reviews Molecular chaperones remodel the in vivo folding energy landscape The ability of molecular chaperones to interact with nascent or incompletely folded chains so as to favor successful folding and disfavor aggregation is well established. Yet the impact of chaperones on the folding mechanisms and stabilities of their clients is less clear, despite expanding literature on the functions and substrate-omes of several chaperones (e.g., Consider the case of arguably the best-studied chaperone, the E. coli chaperonin GroEL and its partner GroES. On Spatial organization, membranes and compartmentalization Cellular interiors are highly anisotropic with elaborate and physiologically critical architectures. This subcellular organization plays a major role in folding at several levels. For example, the native structures of membrane proteins are tuned to the diverse microenvironments and twodimensional character of membranes. Additionally, membranes are barriers, requiring proteins made on one side but destined to perform their functions extracytoplasmically to be translocated across the membrane, in most cases unfolded. For secretory proteins, passage through cellular compartments is highly choreographed with an assembly line of modifying enzymes, chaperones and transport mechanisms. Compartmentalization also opens up the possibility of chemical gradients, for example pH or oxidizing potential. Taken together, the spatial organization and compartmentalization of the cellular environment enable folding to occur in temporally optimized steps. Bacterial proteins destined for the periplasm or the cell exterior can be secreted across membranes either folded (e.g., by the Tat system) or unfolded (e.g., by the Sec system) Proteins that translocate through the Sec channel in bacteria and eukaryotes are greeted by an array of chaperones and modifying enzymes that alter the folding energy landscape. In addition, they move from an ATPrich, reducing environment to one that is ATP-poor and oxidizing as they enter either the bacterial periplasm, mitochondrial intermembrane space (IMS) or the eukaryotic ER. We refer interested readers to recent reviews of protein folding in the ER Exposure of nascent chains to the oxidizing environment of the periplasm or ER lumen enables step-wise disulfide bond formation, fixing the topology of secretory proteins. Not surprisingly, the timing and specificity of disulfide bond formation is integral to their in vivo folding, and this issue has been widely studied in eukaryotic proteins Many proteins that fold in the ER lumen are large with complicated topologies including multiple domains, disulfides, and glyosylation sites, which play roles in specialized in vivo folding mechanisms. A recent study on the influenza membrane glycoprotein neuraminidase Promising new methods for the study of in vivo protein folding The Holy Grail in studies of protein folding in the cell is to directly observe a protein of interest (POI) in intact cells and to characterize its folding, both thermodynamically and kinetically, in situ. Not surprisingly, this has proven exceedingly difficult. Several years ago, Ghaemmaghami and Oas took advantage of E. coli's urea tolerance to perform in-cell urea titrations of the l repressor headpiece utilizing a novel hydrogen exchange/ mass spectrometry method to assess stability Exciting recent work from the Gruebele lab combines temperature-jump perturbation methods with fast relaxation imaging (FReI) to interrogate the in vivo folding landscape of a POI, here a temperature-sensitive variant of phosphoglycerate kinase (tsPGK) In-cell NMR, recently reviewed by Pielak et al. [70] and by Ito and Selenko [71], is a potentially powerful approach to study proteins in vivo and to gain insight into their stability and folding mechanisms. Unfortunately, many folded proteins fail to show measurable NMR spectra in the cellular environment, most likely because of hindered rotational diffusion. Despite the inherent obstacles, the Shirakawa group has had impressive success applying NMR to small proteins in eukaryotic cells Clever use of split reporters, in which folding of the POI is coupled to successful binding and folding of two pieces of 36 Folding and Binding Figure 2 Monitoring protein folding kinetics in a living cell using fast relaxation imaging (FReI) These approaches to in-cell stability use a single flanking reporter and are clearly powerful as selections for enhanced stability. However, they are end-point assays based on the proteolytic lability of the fusion construct when the POI is unstable and therefore cannot readily yield an estimate of folding free energy. A related in vivo screen flanks the POI between a DNA-binding domain and a transcriptional activation domai

Probing the folding pathway of a β-clam protein with single-tryptophan constructs

Background: Cellular retinoic acid binding protein I (CRABPI) is a small, predominantly β-sheet protein with a simple architecture and no disulfides or cofactors. Folding of mutants containing only one of the three native tryptophans has been examined using stopped-flow fluorescence and circular dichroism at multiple wavelengths.Results: Within 10 ms, the tryptophan fluorescence of all three mutants shows a blue shift, and stopped-flow circular dichroism shows significant secondary structure content. The local environment of Trp7, a completely buried residue located near the intersection of the N and C termini, develops on a 100 ms time scale. Spectral signatures of the other two tryptophan residues (87 and 109) become native-like in a 1 s kinetic phase.Conclusions:Formation of the native β structure of CRABPI is initiated by rapid hydrophobic collapse, during which local segments of chain adopt significant secondary structure. Subsequently, transient yet specific interactions of amino acid residues restrict the arrangement of the chain topology and initiate long-range associations such as the docking of the N and C termini. The development of native tertiary environments, including the specific packing of the β-sheet sidechains, occurs in a final, highly cooperative step simultaneous with stable interstrand hydrogen bonding

An interdomain sector mediating allostery in Hsp70 molecular chaperones

The Hsp70 family of molecular chaperones provides a well defined and experimentally powerful model system for understanding allosteric coupling between different protein domains.New extensions to the statistical coupling analysis (SCA) method permit identification of a group of co-evolving amino-acid positions—a sector—in the Hsp70 that is associated with allosteric function.Literature-based and new experimental studies support the notion that the protein sector identified through SCA underlies the allosteric mechanism of Hsp70.This work extends the concept of protein sectors by showing that two non-homologous protein domains can share a single sector when the underlying biological function is defined by the coupled activity of the two domains

Individual and Collective Contributions of Chaperoning and Degradation to Protein Homeostasis in E. coli

SummaryThe folding fate of a protein in vivo is determined by the interplay between a protein’s folding energy landscape and the actions of the proteostasis network, including molecular chaperones and degradation enzymes. The mechanisms of individual components of the E. coli proteostasis network have been studied extensively, but much less is known about how they function as a system. We used an integrated experimental and computational approach to quantitatively analyze the folding outcomes (native folding versus aggregation versus degradation) of three test proteins biosynthesized in E. coli under a variety of conditions. Overexpression of the entire proteostasis network benefited all three test proteins, but the effect of upregulating individual chaperones or the major degradation enzyme, Lon, varied for proteins with different biophysical properties. In sum, the impact of the E. coli proteostasis network is a consequence of concerted action by the Hsp70 system (DnaK/DnaJ/GrpE), the Hsp60 system (GroEL/GroES), and Lon

ATPase Subdomain IA Is a Mediator of Interdomain Allostery in Hsp70 Molecular Chaperones

The versatile functions of the heat shock protein 70 (Hsp70) family of molecular chaperones rely on allosteric interactions between their nucleotide-binding and substrate-binding domains, NBD and SBD. Understanding the mechanism of interdomain allostery is essential to rational design of Hsp70 modulators. Yet, despite significant progress in recent years, how the two Hsp70 domains regulate each other's activity remains elusive. Covariance data from experiments and computations emerged in recent years as valuable sources of information towards gaining insights into the molecular events that mediate allostery. In the present study, conservation and covariance properties derived from both sequence and structural dynamics data are integrated with results from Perturbation Response Scanning and in vivo functional assays, so as to establish the dynamical basis of interdomain signal transduction in Hsp70s. Our study highlights the critical roles of SBD residues D481 and T417 in mediating the coupled motions of the two domains, as well as that of G506 in enabling the movements of the α-helical lid with respect to the β-sandwich. It also draws attention to the distinctive role of the NBD subdomains: Subdomain IA acts as a key mediator of signal transduction between the ATP- and substrate-binding sites, this function being achieved by a cascade of interactions predominantly involving conserved residues such as V139, D148, R167 and K155. Subdomain IIA, on the other hand, is distinguished by strong coevolutionary signals (with the SBD) exhibited by a series of residues (D211, E217, L219, T383) implicated in DnaJ recognition. The occurrence of coevolving residues at the DnaJ recognition region parallels the behavior recently observed at the nucleotide-exchange-factor recognition region of subdomain IIB. These findings suggest that Hsp70 tends to adapt to co-chaperone recognition and activity via coevolving residues, whereas interdomain allostery, critical to chaperoning, is robustly enabled by conserved interactions. © 2014 General et al

Role of Hsp70 ATPase Domain Intrinsic Dynamics and Sequence Evolution in Enabling its Functional Interactions with NEFs

Catalysis of ADP-ATP exchange by nucleotide exchange factors (NEFs) is central to the activity of Hsp70 molecular chaperones. Yet, the mechanism of interaction of this family of chaperones with NEFs is not well understood in the context of the sequence evolution and structural dynamics of Hsp70 ATPase domains. We studied the interactions of Hsp70 ATPase domains with four different NEFs on the basis of the evolutionary trace and co-evolution of the ATPase domain sequence, combined with elastic network modeling of the collective dynamics of the complexes. Our study reveals a subtle balance between the intrinsic (to the ATPase domain) and specific (to interactions with NEFs) mechanisms shared by the four complexes. Two classes of key residues are distinguished in the Hsp70 ATPase domain: (i) highly conserved residues, involved in nucleotide binding, which mediate, via a global hinge-bending, the ATPase domain opening irrespective of NEF binding, and (ii) not-conserved but co-evolved and highly mobile residues, engaged in specific interactions with NEFs (e.g., N57, R258, R262, E283, D285). The observed interplay between these respective intrinsic (pre-existing, structure-encoded) and specific (co-evolved, sequence-dependent) interactions provides us with insights into the allosteric dynamics and functional evolution of the modular Hsp70 ATPase domain

Finding the Fittest Fold: Using the Evolutionary Record to Design New Proteins

For many years, the holy grail of protein engineering has been the design of artificial amino acid sequences that fold into stable proteins with desired functions. In the current issue of Nature, two papers from the Ranganathan group (Russ et al., 2005; Socolich et al., 2005) report remarkable success in the design of artificial WW domains. Their method, termed statistical coupling analysis (Lockless and Ranganathan, 1999), does not use structural or physicochemical information but instead extracts information about essential patterns of amino acids from the evolutionary record