Piecewise All-Atom SMD Simulations Reveal Key Secondary Structures in Luciferase Unfolding Pathway

Although the folding of single-domain proteins is well characterized theoretically and experimentally, the folding of large multidomain proteins is less well known. Firefly luciferase, a 550 residue three-domain protein, has been commonly used as a substrate to study chaperone reactions and as a model system for the study of folding of long polypeptide chains, including related phenomena such as cotranslational folding. Despite being characterized by various experimental techniques, the atomic-level contributions of various secondary structures of luciferase to its fold’s mechanical stability remain unknown. Here, we developed a piecewise approach for all-atom steered molecular dynamics simulations to examine specific secondary structures that resist mechanical unfolding while minimizing the amount of computational resources required by the large water box of standard all-atom steered molecular dynamics simulations. We validated the robustness of this approach with a small NI3C protein and used our approach to elucidate the specific secondary structures that provide the largest contributions to luciferase mechanostability. In doing so, we show that piecewise all-atom steered molecular dynamics simulations can provide novel atomic resolution details regarding mechanostability and can serve as a platform for novel mutagenesis studies as well as a point for comparison with high-resolution force spectroscopy experiments.     

Evolution rescues folding of human immunodeficiency virus-1 envelope glycoprotein GP120 lacking a conserved disulfide bond

The majority of eukaryotic secretory and membrane proteins contain disulfide bonds, which are strongly conserved within protein families because of their crucial role in folding or function. The exact role of these disulfide bonds during folding is unclear. Using virus-driven evolution we generated a viral glycoprotein variant, which is functional despite the lack of an absolutely conserved disulfide bond that links two antiparallel β-strands in a six-stranded β-barrel. Molecular dynamics simulations revealed that improved hydrogen bonding and side chain packing led to stabilization of the β-barrel fold, implying that β-sheet preference codirects glycoprotein folding in vivo. Our results show that the interactions between two β-strands that are important for the formation and/or integrity of the β-barrel can be supported by either a disulfide bond or β-sheet favoring residues.     

ATP‐independent molecular chaperone activity generated under reducing conditions

Molecular chaperones are essential to maintain proteostasis. While the functions of intracellular molecular chaperones that oversee protein synthesis, folding and aggregation, are established, those specialized to work in the extracellular environment are less understood. Extracellular proteins reside in a considerably more oxidizing milieu than cytoplasmic proteins and are stabilized by abundant disulfide bonds. Hence, extracellular proteins are potentially destabilized and sensitive to aggregation under reducing conditions. We combine biochemical and mass spectrometry experiments and elucidate that the molecular chaperone functions of the extracellular protein domain Bri2 BRICHOS only appear under reducing conditions, through the assembly of monomers into large polydisperse oligomers by an intra‐ to intermolecular disulfide bond relay mechanism. Chaperone‐active assemblies of the Bri2 BRICHOS domain are efficiently generated by physiological thiol‐containing compounds and proteins, and appear in parallel with reduction‐induced aggregation of extracellular proteins. Our results give insights into how potent chaperone activity can be generated from inactive precursors under conditions that are destabilizing to most extracellular proteins and thereby support protein stability/folding in the extracellular space.SignificanceChaperones are essential to cells as they counteract toxic consequences of protein misfolding particularly under stress conditions. Our work describes a novel activation mechanism of an extracellular molecular chaperone domain, called Bri2 BRICHOS. This mechanism is based on reducing conditions that initiate small subunits to assemble into large oligomers via a disulfide relay mechanism. Activated Bri2 BRICHOS inhibits reduction‐induced aggregation of extracellular proteins and could be a means to boost proteostasis in the extracellular environment upon reductive stress.     

Expression, folding, and characterization of small proteins with increasing disulfide complexity by a pT7-7-derived phagemid

The expression, folding, and characterization of a series of small proteins with increasingly complex disulfide bond patterns were characterized. A phagemid was prepared from the pT7-7 plasmid to facilitate mutagenic studies with these proteins. cDNAs coding for bovine, rat, and human prolactin; human growth hormone; and bovine alpha-lactalbumin were amplified by PCR using primers that inserted restriction sites at the 5' and 3' ends and reduced the coding sequence to the mature methionyl protein with bacterially preferred codons in the 5' region. The expressed proteins were folded and oxidized by methods that allowed disulfide bond formation to occur either during or following folding. The effectiveness of the folding procedures was determined for each protein by electrophoresis, absorption spectroscopy, and functional studies. The redox conditions required for folding functional proteins varied as the number of disulfide bonds per unit molecular weight increased. Human growth hormone, 22 kDa; human prolactin, 23 kDa; and bovine prolactin, 23 kDa, contain two, three, and three disulfides, respectively, and are folded correctly by air oxidation performed during renaturation under alkaline conditions. Proper disulfide bond formation of rat prolactin, 23 kDa, containing three disulfide bonds required the addition of a reducing agent at the initiation of renaturation. Bovine alpha-lactalbumin, 14 kDa with four disulfide bonds, required complete renaturation prior to the removal of a reducing agent. SDS-gel electrophoresis under nonreducing conditions provided information regarding the proper folding of these proteins. The absorption of 250-nm light by disulfide bonds also provided information regarding the proper folding of rat prolactin and bovine alpha-lactalbumin.     

Structural Conversion of Aβ17–42 Peptides from Disordered Oligomers to U-Shape Protofilaments via Multiple Kinetic Pathways

Discovering the mechanisms by which proteins aggregate into fibrils is an essential first step in understanding the molecular level processes underlying neurodegenerative diseases such as Alzheimer’s and Parkinson''s. The goal of this work is to provide insights into the structural changes that characterize the kinetic pathways by which amyloid-β peptides convert from monomers to oligomers to fibrils. By applying discontinuous molecular dynamics simulations to PRIME20, a force field designed to capture the chemical and physical aspects of protein aggregation, we have been able to trace out the entire aggregation process for a system containing 8 Aβ17–42 peptides. We uncovered two fibrillization mechanisms that govern the structural conversion of Aβ17–42 peptides from disordered oligomers into protofilaments. The first mechanism is monomeric conversion templated by a U-shape oligomeric nucleus into U-shape protofilament. The second mechanism involves a long-lived and on-pathway metastable oligomer with S-shape chains, having a C-terminal turn, en route to the final U-shape protofilament. Oligomers with this C-terminal turn have been regarded in recent experiments as a major contributing element to cell toxicity in Alzheimer’s disease. The internal structures of the U-shape protofilaments from our PRIME20/DMD simulation agree well with those from solid state NMR experiments. The approach presented here offers a simple molecular-level framework to describe protein aggregation in general and to visualize the kinetic evolution of a putative toxic element in Alzheimer’s disease in particular.     

Protein quality control in the ER: The recognition of misfolded proteins

The mechanism, in molecular terms of protein quality control, specifically of how the cell recognizes and discriminates misfolded proteins, remains a challenge. In the secretory pathway the folding status of glycoproteins passing through the endoplasmic reticulum is marked by the composition of the N-glycan. The different glycoforms are recognized by specialized lectins. The folding sensor UGGT acts as an unusual molecular chaperone and covalently modifies the Man9 N-glycan of a misfolded protein by adding a glucose moiety and converts it to Glc1Man9 that rebinds the lectin calnexin. However, further links between the folding status of a glycoprotein and the composition of the N-glycan are unclear. There is little unequivocal evidence for other proteins in the ER recognizing the N-glycan and also acting as molecular chaperones. Nevertheless, based upon a few examples, we suggest that this function is carried out by individual proteins in several different complexes. Thus, calnexin binds the protein disulfide isomerase ERp57, that acts upon Glc1Man9 glycoproteins. In another example the protein disulfide isomerase ERdj5 binds specifically to EDEM (which is probably a mannosidase) and a lectin OS9, and reduces the disulfide bonds of bound glycoproteins destined for ERAD. Thus the glycan recognition is performed by a lectin and the chaperone function performed by a specific partner protein that can recognize misfolded proteins. We predict that this will be a common arrangement of proteins in the ER and that members of protein foldase families such as PDI and PPI will bind specifically to lectins in the ER. Molecular chaperones BiP and GRp94 will assist in the folding of proteins bound in these complexes as well as in the folding of non-glycoproteins.     

Rheostat Re-Wired: Alternative Hypotheses for the Control of Thioredoxin Reduction Potentials

Thioredoxins are small soluble proteins that contain a redox-active disulfide (CXXC). These disulfides are tuned to oxidizing or reducing potentials depending on the function of the thioredoxin within the cell. The mechanism by which the potential is tuned has been controversial, with two main hypotheses: first, that redox potential (E_m) is specifically governed by a molecular ‘rheostat’—the XX amino acids, which influence the Cys pK_a values, and thereby, E_m; and second, the overall thermodynamics of protein folding stability regulates the potential. Here, we use protein film voltammetry (PFV) to measure the pH dependence of the redox potentials of a series of wild-type and mutant archaeal Trxs, PFV and glutathionine-equilibrium to corroborate the measured potentials, the fluorescence probe BADAN to measure pK_a values, guanidinium-based denaturation to measure protein unfolding, and X-ray crystallography to provide a structural basis for our functional analyses. We find that when these archaeal thioredoxins are probed directly using PFV, both the high and low potential thioredoxins display consistent 2H⁺:2e^- coupling over a physiological pH range, in conflict with the conventional ‘rheostat’ model. Instead, folding measurements reveals an excellent correlation to reduction potentials, supporting the second hypothesis and revealing the molecular mechanism of reduction potential control in the ubiquitous Trx family.     

What can disulfide bonds tell us about protein energetics, function and folding: simulations and bioninformatics analysis

We study the impact of disulfide bonds on protein stability and folding. Using lattice model simulations, we show that formation of a disulfide bond stabilizes a protein to an extent that depends on the distance along the chain between linked cysteine residues. However, the impact of disulfide bonds on folding kinetics varies broadly, from acceleration when disulfides are introduced in or close to the folding nucleus, to slowing when disulfides are introduced outside the nucleus. Having established the effect of disulfide bonds on stability, we study the correlation between the number of disulfide bonds and the composition of certain amino acid classes with the goal to use it as a statistical probe into factors that contribute to stability of proteins. We find that the number of disulfides is negatively correlated with aliphatic hydrophobic but not aromatic content. It is surprising that we observe a strong correlation of disulfide content with polar (Q,S,T,N) amino acid content and a strong negative correlation with charged (E,D,K,R) content. These findings provide insights into factors that determine protein stability and principles of protein design as well as possible relations of disulfide bonds and protein function.     

DsbG, a protein disulfide isomerase with chaperone activity

DsbG, a protein disulfide isomerase present in the periplasm of Escherichia coli, is shown to function as a molecular chaperone. Stoichiometric amounts of DsbG are sufficient to prevent the thermal aggregation of two classical chaperone substrate proteins, citrate synthase and luciferase. DsbG was also shown to interact with refolding intermediates of chemically denatured citrate synthase and prevents their aggregation in vitro. Citrate synthase reactivation experiments in the presence of DsbG suggest that DsbG binds with high affinity to early unstructured protein folding intermediates. DsbG is one of the first periplasmic proteins shown to have general chaperone activity. This ability to chaperone protein folding is likely to increase the effectiveness of DsbG as a protein disulfide isomerase.     

Simulation and experiment conspire to reveal cryptic intermediates and a slide from the nucleation-condensation to framework mechanism of folding

There is a change from three-state to two-state kinetics of folding across the homeodomain superfamily of proteins as the mechanism slides from framework to nucleation-condensation. The tendency for framework folding in this family correlates with inherent helical propensity. The cellular myeloblastis protein (c-Myb) falls in the mechanistic transition region. An earlier, preliminary report of protein engineering experiments and molecular dynamics simulations (MD) showed that the folding mechanism for this protein has aspects of both the nucleation-condensation and framework models. In the more in-depth analysis of the MD trajectories presented here, we find that folding may be attributed to both of these mechanisms in different regions of the protein. The folding of the loop, middle helix, and turn is best described by nucleation-condensation, whereas folding of the N and C-terminal helices may be described by the framework model. Experimentally, c-Myb folds by apparent two-state kinetics, but the MD simulations predict that the kinetics hide a high-energy intermediate. We stabilized this hypothetical folding intermediate by deleting a residue (P174) in the loop between its second and third helices, and the mutant intermediate is long-lived in the simulations. Equilibrium and kinetic experiments demonstrate that folding of the DeltaP174 mutant is indeed three-state. The presence and shape of the intermediate observed in the simulations were confirmed by small angle X-ray scattering experiments.     

Disulfide bond formation network in the three biological kingdoms, bacteria, fungi and mammals

Almost all organisms, from bacteria to humans, possess catalytic systems that promote disulfide bond formation-coupled protein folding, i.e. oxidative protein folding. These systems are necessary for the biosynthesis of many secretory and membrane proteins, such as antibodies, major histocompatibility complex molecules, growth factors, and insulin. Over the last decade, structural studies have made striking progress in this field of research, identifying how oxidative systems operate in a specific and regulated manner to maintain redox and protein homeostasis within cells. Interestingly, more and more novel catalysts that promote disulfide bond formation have been discovered in mammals, suggesting that the oxidative protein folding network is even more complicated in higher eukaryotes than previously thought. This review highlights the physiological roles and molecular bases of the disulfide bond formation pathways that have evolved in the bacterial periplasm and the endoplasmic reticulum of fungi and mammals. Accumulating knowledge about disulfide bond formation networks widely distributed throughout the biological kingdom has significantly advanced our understanding of the cellular mechanisms dedicated to protein quality control.     

Oxidative protein folding in the plant endoplasmic reticulum

For most of the proteins synthesized in the endoplasmic reticulum (ER), disulfide bond formation accompanies protein folding in a process called oxidative folding. Oxidative folding is catalyzed by a number of enzymes, including the family of protein disulfide isomerases (PDIs), as well as other proteins that supply oxidizing equivalents to PDI family proteins, like ER oxidoreductin 1 (Ero1). Oxidative protein folding in the ER is a basic vital function, and understanding its molecular mechanism is critical for the application of plants as protein production tools. Here, I review the recent research and progress related to the enzymes involved in oxidative folding in the plant ER. Firstly, nine groups of plant PDI family proteins are introduced. Next, the enzymatic properties of plant Ero1 are described. Finally, the cooperative folding by multiple PDI family proteins and Ero1 is described.     

Polymer Uncrossing and Knotting in Protein Folding,and Their Role in Minimal Folding Pathways

We introduce a method for calculating the extent to which chain non-crossing is important in the most efficient, optimal trajectories or pathways for a protein to fold. This involves recording all unphysical crossing events of a ghost chain, and calculating the minimal uncrossing cost that would have been required to avoid such events. A depth-first tree search algorithm is applied to find minimal transformations to fold , , , and knotted proteins. In all cases, the extra uncrossing/non-crossing distance is a small fraction of the total distance travelled by a ghost chain. Different structural classes may be distinguished by the amount of extra uncrossing distance, and the effectiveness of such discrimination is compared with other order parameters. It was seen that non-crossing distance over chain length provided the best discrimination between structural and kinetic classes. The scaling of non-crossing distance with chain length implies an inevitable crossover to entanglement-dominated folding mechanisms for sufficiently long chains. We further quantify the minimal folding pathways by collecting the sequence of uncrossing moves, which generally involve leg, loop, and elbow-like uncrossing moves, and rendering the collection of these moves over the unfolded ensemble as a multiple-transformation “alignment”. The consensus minimal pathway is constructed and shown schematically for representative cases of an , , and knotted protein. An overlap parameter is defined between pathways; we find that proteins have minimal overlap indicating diverse folding pathways, knotted proteins are highly constrained to follow a dominant pathway, and proteins are somewhere in between. Thus we have shown how topological chain constraints can induce dominant pathway mechanisms in protein folding.     

Influence of molecular and chemical chaperones on protein folding

Protein folding inside the cell involves the Participation of accessory components known as molecular chaperones. In addition to their active participation in the folding process, molecular chaperones serve as a type of ‘quality control system’, recognizing, retaining and targeting misfolded proteins for their eventual degradation. It is now known that a number of human diseases arise as a consequence of specific point mutations or deletions within genes encoding essential proteins. In many cases these mutations/deletions are not so sever as to totally destroy the biological activity of the particular gene product. Rather, the mutations often result in only subtle folding abnormalities which lead to the newly synthesized protein being retained at the endoplasmic reticulum by the actions of the cellylar quality control system. In this short review article we discuss our recent studies showing that the protein folding defect associated with the most common mutation in patients with cystic fibriosis can be overcome by a novel strategy. As shown in the paper by Brown et al in this issue (Brown et al 1996), a number of different low molecular weight compounds, all known to stabilize proteins in their native conformation, are effective in rescuing the processing defect of the mutant cystic fibrosis transmembrane conductance regulator protein. We then discuss how these same compounds, which we now call chemical chaperones, also may prove to be effective in correcting a number of other protein folding abnormalities which constitute the underlying basis of a large number of different human diseases.     

Dynamics of Bcl-xL in Water and Membrane: Molecular Simulations

The Bcl2 family of proteins is capable of switching the apoptotic machinery by directly controlling the release of apoptotic factors from the mitochondrial outer membrane. They have ‘pro’ and ‘anti’-apoptotic subgroups of proteins which antagonize each other’s function; however a detailed atomistic understanding of their mechanisms based on the dynamical events, particularly in the membrane, is lacking. Using molecular dynamics simulations totaling 1.6µs we outline the major differences between the conformational dynamics in water and in membrane. Using implicit models of solvent and membrane, the simulated results reveal a picture that is in agreement with the ‘hit-and run’ concept which states that BH3-only peptides displace the tail (which acts as a pseudo substrate of the protein itself) from its binding pocket; this helps the membrane association of the protein after which the BH3 peptide becomes free. From simulations, Bcl-xL appears to be auto-inhibited by its C-terminal tail that embeds into and covers the hydrophobic binding pocket. However the tail is unable to energetically compete with BH3-peptides in water. In contrast, in the membrane, neither the tail nor the BH3-peptides are stable in the binding pocket and appear to be easily dissociated off as the pocket expands in response to the hydrophobic environment. This renders the binding pocket large and open, thus receptive to interactions with other protein partners. Principal components of the motions are dramatically different in the aqueous and in the membrane environments and provide clues regarding the conformational transitions that Bcl-xL undergoes in the membrane, in agreement with the biochemical data.     

Understanding the Folding-Function Tradeoff in Proteins

When an amino-acid sequence cannot be optimized for both folding and function, folding can get compromised in favor of function. To understand this tradeoff better, we devise a novel method for extracting the “function-less” folding-motif of a protein fold from a set of structurally similar but functionally diverse proteins. We then obtain the β-trefoil folding-motif, and study its folding using structure-based models and molecular dynamics simulations. CompariA protein sequence serves two purpson with the folding of wild-type β-trefoil proteins shows that function affects folding in two ways: In the slower folding interleukin-1β, binding sites make the fold more complex, increase contact order and slow folding. In the faster folding hisactophilin, residues which could have been part of the folding-motif are used for function. This reduces the density of native contacts in functional regions and increases folding rate. The folding-motif helps identify subtle structural deviations which perturb folding. These may then be used for functional annotation. Further, the folding-motif could potentially be used as a first step in the sequence design of function-less scaffold proteins. Desired function can then be engineered into these scaffolds.     

Structure of the Non-Catalytic Domain of the Protein Disulfide Isomerase-Related Protein (PDIR) Reveals Function in Protein Binding

Protein disulfide isomerases comprise a large family of enzymes responsible for catalyzing the proper oxidation and folding of newly synthesized proteins in the endoplasmic reticulum (ER). Protein disulfide isomerase-related (PDIR) protein (also known as PDIA5) is a specialized member that participates in the folding of α₁-antitrypsin and N-linked glycoproteins. Here, the crystal structure of the non-catalytic domain of PDIR was determined to 1.5 Å resolution. The structure adopts a thioredoxin-like fold stabilized by a structural disulfide bridge with a positively charged binding surface for interactions with the ER chaperones, calreticulin and ERp72. Crystal contacts between molecules potentially mimic the interactions of PDIR with misfolded substrate proteins. The results suggest that the non-catalytic domain of PDIR plays a key role in the recognition of protein partners and substrates.     

Molecular simulations of cotranslational protein folding: fragment stabilities, folding cooperativity, and trapping in the ribosome

Although molecular simulation methods have yielded valuable insights into mechanistic aspects of protein refolding in vitro, they have up to now not been used to model the folding of proteins as they are actually synthesized by the ribosome. To address this issue, we report here simulation studies of three model proteins: chymotrypsin inhibitor 2 (CI2), barnase, and Semliki forest virus protein (SFVP), and directly compare their folding during ribosome-mediated synthesis with their refolding from random, denatured conformations. To calibrate the methodology, simulations are first compared with in vitro data on the folding stabilities of N-terminal fragments of CI2 and barnase; the simulations reproduce the fact that both the stability and thermal folding cooperativity increase as fragments increase in length. Coupled simulations of synthesis and folding for the same two proteins are then described, showing that both fold essentially post-translationally, with mechanisms effectively identical to those for refolding. In both cases, confinement of the nascent polypeptide chain within the ribosome tunnel does not appear to promote significant formation of native structure during synthesis; there are however clear indications that the formation of structure within the nascent chain is sensitive to location within the ribosome tunnel, being subject to both gain and loss as the chain lengthens. Interestingly, simulations in which CI2 is artificially stabilized show a pronounced tendency to become trapped within the tunnel in partially folded conformations: non-cooperative folding, therefore, appears in the simulations to exert a detrimental effect on the rate at which fully folded conformations are formed. Finally, simulations of the two-domain protease module of SFVP, which experimentally folds cotranslationally, indicate that for multi-domain proteins, ribosome-mediated folding may follow different pathways from those taken during refolding. Taken together, these studies provide a first step toward developing more realistic methods for simulating protein folding as it occurs in vivo.