The entropic force generated by intrinsically disordered segments tunes protein function.

Protein structures are dynamic and can explore a large conformational landscape1,2. Only some of these structural substates are important for protein function (such as ligand binding, catalysis and regulation)3-5. How evolution shapes the structural ensemble to optimize a specific function is poorly understood3,4. One of the constraints on the evolution of proteins is the stability of the folded 'native' state. Despite this, 44% of the human proteome contains intrinsically disordered peptide segments greater than 30 residues in length6, the majority of which have no known function7-9. Here we show that the entropic force produced by an intrinsically disordered carboxy terminus (ID-tail) shifts the conformational ensemble of human UDP-α-D-glucose-6-dehydrogenase (UGDH) towards a substate with a high affinity for an allosteric inhibitor. The function of the ID-tail does not depend on its sequence or chemical composition. Instead, the affinity enhancement can be accurately predicted based on the length of the intrinsically disordered segment, and is consistent with the entropic force generated by an unstructured peptide attached to the protein surface10-13. Our data show that the unfolded state of the ID-tail rectifies the dynamics and structure of UGDH to favour inhibitor binding. Because this entropic rectifier does not have any sequence or structural constraints, it is an easily acquired adaptation. This model implies that evolution selects for disordered segments to tune the energy landscape of proteins, which may explain the persistence of intrinsic disorder in the proteome.

Function of a viral genome packaging motor from bacteriophage T4 is insensitive to DNA sequence.

Many viruses employ ATP-powered motors during assembly to translocate DNA into procapsid shells. Previous reports raise the question if motor function is modulated by substrate DNA sequence: (i) the phage T4 motor exhibits large translocation rate fluctuations and pauses and slips; (ii) evidence suggests that the phage phi29 motor contacts DNA bases during translocation; and (iii) one theoretical model, the 'B-A scrunchworm', predicts that 'A-philic' sequences that transition more easily to A-form would alter motor function. Here, we use single-molecule optical tweezers measurements to compare translocation of phage, plasmid, and synthetic A-philic, GC rich sequences by the T4 motor. We observed no significant differences in motor velocities, even with A-philic sequences predicted to show higher translocation rate at high applied force. We also observed no significant changes in motor pausing and only modest changes in slipping. To more generally test for sequence dependence, we conducted correlation analyses across pairs of packaging events. No significant correlations in packaging rate, pausing or slipping versus sequence position were detected across repeated measurements with several different DNA sequences. These studies suggest that viral genome packaging is insensitive to DNA sequence and fluctuations in packaging motor velocity, pausing and slipping are primarily stochastic temporal events.

DNA Conformational Changes Play a Force-Generating Role during Bacteriophage Genome Packaging.

Motors that move DNA, or that move along DNA, play essential roles in DNA replication, transcription, recombination, and chromosome segregation. The mechanisms by which these DNA translocases operate remain largely unknown. Some double-stranded DNA (dsDNA) viruses use an ATP-dependent motor to drive DNA into preformed capsids. These include several human pathogens as well as dsDNA bacteriophages-viruses that infect bacteria. We previously proposed that DNA is not a passive substrate of bacteriophage packaging motors but is instead an active component of the machinery. We carried out computational studies on dsDNA in the channels of viral portal proteins, and they reveal DNA conformational changes consistent with that hypothesis. dsDNA becomes longer ("stretched") in regions of high negative electrostatic potential and shorter ("scrunched") in regions of high positive potential. These results suggest a mechanism that electrostatically couples the energy released by ATP hydrolysis to DNA translocation: The chemical cycle of ATP binding, hydrolysis, and product release drives a cycle of protein conformational changes. This produces changes in the electrostatic potential in the channel through the portal, and these drive cyclic changes in the length of dsDNA as the phosphate groups respond to the protein's electrostatic potential. The DNA motions are captured by a coordinated protein-DNA grip-and-release cycle to produce DNA translocation. In short, the ATPase, portal, and dsDNA work synergistically to promote genome packaging.

History of the ribosome and the origin of translation.

We present a molecular-level model for the origin and evolution of the translation system, using a 3D comparative method. In this model, the ribosome evolved by accretion, recursively adding expansion segments, iteratively growing, subsuming, and freezing the rRNA. Functions of expansion segments in the ancestral ribosome are assigned by correspondence with their functions in the extant ribosome. The model explains the evolution of the large ribosomal subunit, the small ribosomal subunit, tRNA, and mRNA. Prokaryotic ribosomes evolved in six phases, sequentially acquiring capabilities for RNA folding, catalysis, subunit association, correlated evolution, decoding, energy-driven translocation, and surface proteinization. Two additional phases exclusive to eukaryotes led to tentacle-like rRNA expansions. In this model, ribosomal proteinization was a driving force for the broad adoption of proteins in other biological processes. The exit tunnel was clearly a central theme of all phases of ribosomal evolution and was continuously extended and rigidified. In the primitive noncoding ribosome, proto-mRNA and the small ribosomal subunit acted as cofactors, positioning the activated ends of tRNAs within the peptidyl transferase center. This association linked the evolution of the large and small ribosomal subunits, proto-mRNA, and tRNA.

Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy.

The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures.

Evolution of the ribosome at atomic resolution.

The origins and evolution of the ribosome, 3-4 billion years ago, remain imprinted in the biochemistry of extant life and in the structure of the ribosome. Processes of ribosomal RNA (rRNA) expansion can be "observed" by comparing 3D rRNA structures of bacteria (small), yeast (medium), and metazoans (large). rRNA size correlates well with species complexity. Differences in ribosomes across species reveal that rRNA expansion segments have been added to rRNAs without perturbing the preexisting core. Here we show that rRNA growth occurs by a limited number of processes that include inserting a branch helix onto a preexisting trunk helix and elongation of a helix. rRNA expansions can leave distinctive atomic resolution fingerprints, which we call "insertion fingerprints." Observation of insertion fingerprints in the ribosomal common core allows identification of probable ancestral expansion segments. Conceptually reversing these expansions allows extrapolation backward in time to generate models of primordial ribosomes. The approach presented here provides insight to the structure of pre-last universal common ancestor rRNAs and the subsequent expansions that shaped the peptidyl transferase center and the conserved core. We infer distinct phases of ribosomal evolution through which ribosomal particles evolve, acquiring coding and translocation, and extending and elaborating the exit tunnel.

The scrunchworm hypothesis: transitions between A-DNA and B-DNA provide the driving force for genome packaging in double-stranded DNA bacteriophages.

Double-stranded DNA bacteriophages have motors that drive the genome into preformed capsids, using the energy released by hydrolysis of ATP to overcome the forces opposing DNA packaging. Viral packaging motors are the strongest of all biological motors, but it is not known how they generate these forces. Several models for the process of mechanochemical force generation have been put forward, but there is no consensus on which, if any, of these is correct. All the existing models assume that protein-generated forces drive the DNA forward. The scrunchworm hypothesis proposes that the DNA molecule is the active force-generating core of the motor, not simply a substrate on which the motor operates. The protein components of the motor dehydrate a section of the DNA, converting it from the B form to the A form and shortening it by about 23%. The proteins then rehydrate the DNA, which converts back to the B form. Other regions of the motor grip and release the DNA to capture the shortening-lengthening motions of the B→A→B cycle ("scrunching"), so that DNA is pulled into the motor and pushed forward into the capsid. This DNA-centric mechanism provides a quantitative physical explanation for the magnitude of the forces generated by viral packaging motors. It also provides a simple explanation for the fact that each of the steps in the burst cycle advances the DNA by 2.5 base pairs. The scrunchworm hypothesis is consistent with a large body of published data, and it makes four experimentally testable predictions.

Secondary structure and domain architecture of the 23S and 5S rRNAs.

We present a de novo re-determination of the secondary (2°) structure and domain architecture of the 23S and 5S rRNAs, using 3D structures, determined by X-ray diffraction, as input. In the traditional 2° structure, the center of the 23S rRNA is an extended single strand, which in 3D is seen to be compact and double helical. Accurately assigning nucleotides to helices compels a revision of the 23S rRNA 2° structure. Unlike the traditional 2° structure, the revised 2° structure of the 23S rRNA shows architectural similarity with the 16S rRNA. The revised 2° structure also reveals a clear relationship with the 3D structure and is generalizable to rRNAs of other species from all three domains of life. The 2° structure revision required us to reconsider the domain architecture. We partitioned the 23S rRNA into domains through analysis of molecular interactions, calculations of 2D folding propensities and compactness. The best domain model for the 23S rRNA contains seven domains, not six as previously ascribed. Domain 0 forms the core of the 23S rRNA, to which the other six domains are rooted. Editable 2° structures mapped with various data are provided (http://apollo.chemistry.gatech.edu/RibosomeGallery).

Molecular paleontology: a biochemical model of the ancestral ribosome.

Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates ∼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.

Comment on the letter by A. Ben-Shaul: "entropy, energy, and bending of DNA in viral capsids".

The conformational entropic penalty associated with packaging double-stranded DNA into viral capsids remains an issue of contention. So far, models based on a continuum approximation for DNA have either left the question unexamined, or they have assumed that the entropic penalty is negligible, following an early analysis by Riemer and Bloomfield. In contrast, molecular-dynamics (MD) simulations using bead-and-spring models consistently show a large penalty. A recent letter from Ben-Shaul attempts to reconcile the differences. While the letter makes some valid points, the issue of how to include conformational entropy in the continuum models remains unresolved. In this Comment, I show that the free energy decomposition from continuum models could be brought into line with the decomposition from the MD simulations with two adjustments. First, the entropy from Flory-Huggins theory should be replaced by the estimate of the entropic penalty given in Ben-Shaul's letter, which corresponds closely to that from the MD simulations. Second, the DNA-DNA repulsions are well described by the empirical relationship given by the Cal Tech group, but the strength of these should be reduced by about half, using parameters based on the Rau-Parsegian experiments, rather than treating them as "fitting parameters (tuned) to fit the data from (single molecule pulling) experiments."

Domain III of the T. thermophilus 23S rRNA folds independently to a near-native state.

The three-dimensional structure of the ribosomal large subunit (LSU) reveals a single morphological element, although the 23S rRNA is contained in six secondary structure domains. Based upon maps of inter- and intra-domain interactions and proposed evolutionary pathways of development, we hypothesize that Domain III is a truly independent structural domain of the LSU. Domain III is primarily stabilized by intra-domain interactions, negligibly perturbed by inter-domain interactions, and is not penetrated by ribosomal proteins or other rRNA. We have probed the structure of Domain III rRNA alone and when contained within the intact 23S rRNA using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension), in the absence and presence of magnesium. The combined results support the hypothesis that Domain III alone folds to a near-native state with secondary structure, intra-domain tertiary interactions, and inter-domain interactions that are independent of whether or not it is embedded in the intact 23S rRNA or within the LSU. The data presented support previous suggestions that Domain III was added relatively late in ribosomal evolution.

Biophysical and ultrastructural characterization of adeno-associated virus capsid uncoating and genome release.

We describe biophysical and ultrastructural differences in genome release from adeno-associated virus (AAV) capsids packaging wild-type DNA, recombinant single-stranded DNA (ssDNA), or dimeric, self-complementary DNA (scDNA) genomes. Atomic force microscopy and electron microscopy (EM) revealed that AAV particles release packaged genomes and undergo marked changes in capsid morphology upon heating in physiological buffer (pH 7.2). When different AAV capsids packaging ss/scDNA varying in length from 72 to 123% of wild-type DNA (3.4 to 5.8 kb) were incrementally heated, the proportion of uncoated AAV capsids decreased with genome length as observed by EM. Genome release was further characterized by a fluorimetric assay, which demonstrated that acidic pH and high osmotic pressure suppress genome release from AAV particles. In addition, fluorimetric analysis corroborated an inverse correlation between packaged genome length and the temperature needed to induce uncoating. Surprisingly, scAAV vectors required significantly higher temperatures to uncoat than their ssDNA-packaging counterparts. However, externalization of VP1 N termini appears to be unaffected by packaged genome length or self-complementarity. Further analysis by tungsten-shadowing EM revealed striking differences in the morphologies of ssDNA and scDNA genomes upon release from intact capsids. Computational modeling and molecular dynamics simulations suggest that the unusual thermal stability of scAAV vectors might arise from partial base pairing and optimal organization of packaged scDNA. Our work further defines the biophysical mechanisms underlying adeno-associated virus uncoating and genome release.

Bidentate RNA-magnesium clamps: on the origin of the special role of magnesium in RNA folding.

Magnesium plays a special role in RNA function and folding. Although water is magnesium's most common first-shell ligand, the oxyanions of RNA have significant affinity for magnesium. Here we provide a quantum mechanical description of first-shell RNA-magnesium and DNA-magnesium interactions, demonstrating the unique features that characterize the energetics and geometry of magnesium complexes within large folded RNAs. Our work focuses on bidentate chelation of magnesium by RNA or DNA, where multiple phosphate oxyanions enter the first coordination shell of magnesium. These bidentate RNA clamps of magnesium occur frequently in large RNAs. The results here suggest that magnesium, compared to calcium and sodium, has an enhanced ability to form bidentate clamps with RNA. Bidentate RNA-sodium clamps, in particular, are unstable and spontaneously open. Due to magnesium's size and charge density it binds more intimately than other cations to the oxyanions of RNA, so that magnesium clamps are stabilized not only by electrostatic interactions, but also by charge transfer, polarization, and exchange interactions. These nonelectrostatic components of the binding are quite substantial with the high charge and small interatomic distances within the magnesium complexes, but are less pronounced for calcium due to its larger size, and for sodium due to its smaller charge. Additionally, bidentate RNA clamps of magnesium are more stable than those with DNA. The source of the additional stability of RNA complexes is twofold: there is a slightly attenuated energetic penalty for ring closure in the formation of RNA bidentate chelation complexes and elevated electrostatic interactions between the RNA and cations. In sum, it can be seen why sodium and calcium cannot replicate the structures or energetics of RNA-magnesium complexes.

A model for the structure of satellite tobacco mosaic virus.

Satellite tobacco mosaic virus (STMV) is an icosahedral T=1 single-stranded RNA virus with a genome containing 1058 nucleotides. X-ray crystallography revealed a structure containing 30 double-helical RNA segments, with each helix having nine base pairs and an unpaired nucleotide at the 3' end of each strand. Based on this structure, Larson and McPherson proposed a model of 30 hairpin-loop elements occupying the edges of the icosahedron and connected by single-stranded regions. More recently, Schroeder et al. have combined the results of chemical probing with a novel helix searching algorithm to propose a specific secondary structure for the STMV genome, compatible with the Larson-McPherson model. Here we report an all-atom model of STMV, using the complete protein and RNA sequences and the Schroeder RNA secondary structure. As far as we know, this is the first all-atom model for the complete structure of any virus (100% of the atoms) using the natural genomic sequence.

Molecular dynamics simulations of forced unbending of integrin α(v)ß3.

Integrins may undergo large conformational changes during activation, but the dynamic processes and pathways remain poorly understood. We used molecular dynamics to simulate forced unbending of a complete integrin α(v)ß3 ectodomain in both unliganded and liganded forms. Pulling the head of the integrin readily induced changes in the integrin from a bent to an extended conformation. Pulling at a cyclic RGD ligand bound to the integrin head also extended the integrin, suggesting that force can activate integrins. Interactions at the interfaces between the hybrid and ß tail domains and between the hybrid and epidermal growth factor 4 domains formed the major energy barrier along the unbending pathway, which could be overcome spontaneously in ~1 µs to yield a partially-extended conformation that tended to rebend. By comparison, a fully-extended conformation was stable. A newly-formed coordination between the α(v) Asp457 and the α-genu metal ion might contribute to the stability of the fully-extended conformation. These results reveal the dynamic processes and pathways of integrin conformational changes with atomic details and provide new insights into the structural mechanisms of integrin activation.

Computational screening and design of DNA-linked molecular nanowires.

DNA can be used as a structural component in the process of making conductive polymers called nanowires. Accurate molecular models could lead to a better understanding of how to prepare these types of materials. Here we present a computational tool that allows potential DNA-linked polymer designs to be screened and evaluated. The approach involves an iterative procedure that adjusts the positions of DNA-linked monomers in order to obtain reasonable molecular geometry compatible with normal DNA conformations and with the properties of the polymer being formed. This procedure has been used to evaluate designs already reported experimentally, as well as to suggest a new design based on pyrrylene vinylene (PV) monomers.

Role of DNA-DNA interactions on the structure and thermodynamics of bacteriophages Lambda and P4.

Electrostatic interactions play an important role in both packaging of DNA inside bacteriophages and its release into bacterial cells. While at physiological conditions DNA strands repel each other, the presence of polyvalent cations such as spermine and spermidine in solutions leads to the formation of DNA condensates. In this study, we discuss packaging of DNA into bacteriophages P4 and Lambda under repulsive and attractive conditions using a coarse-grained model of DNA and capsids. Packaging under repulsive conditions leads to the appearance of the coaxial spooling conformations; DNA occupies all available space inside the capsid. Under the attractive potential both packed systems reveal toroidal conformations, leaving the central part of the capsids empty. We also present a detailed thermodynamic analysis of packaging and show that the forces required to pack the genomes in the presence of polyamines are significantly lower than those observed under repulsive conditions. The analysis reveals that in both the repulsive and attractive regimes the entropic penalty of DNA confinement has a significant non-negligible contribution into the total energy of packaging. Additionally we report the results of simulations of DNA condensation inside partially packed Lambda. We found that at low densities DNA behaves as free unconfined polymer and condenses into the toroidal structures; at higher densities rearrangement of the genome into toroids becomes hindered, and condensation results in the formation of non-equilibrium structures. In all cases packaging in a specific conformation occurs as a result of interplay between bending stresses experienced by the confined polymer and interactions between the strands.

Integration host factor (IHF) dictates the structure of polyamine-DNA condensates: implications for the role of IHF in the compaction of bacterial chromatin.

Integration host factor (IHF), a nucleoid-associated protein in bacterial cells, is implicated in a number of chromosomal functions including DNA compaction. IHF binds to all duplex DNA with micromolar affinity and at sequence-specific sites with much higher affinity. IHF is known to induce sharp bends in the helical axis of DNA in both modes of binding, but the role of IHF in controlling DNA condensation within bacterial cells has remained undetermined. Here we demonstrate that IHF influences the morphology of DNA condensed by polyamines in vitro. In the absence of IHF, spermidine and spermine condense DNA primarily into toroidal structures, whereas in the presence of IHF, polyamines condense DNA primarily into rodlike structures. Computer simulations of DNA condensation in the absence and presence of IHF binding lend support to our model in which DNA bending proteins, such as IHF and HU, promote the condensation of DNA into rodlike structures by providing the free energy necessary to bend DNA at the ends of linear bundles of condensed DNA. We propose that a common function of IHF and HU in bacterial cells is to facilitate DNA organization in the nucleoid by the introduction of sharp bends in chromosomal DNA.

Viral assembly: a molecular modeling perspective.

Icosahedral viruses are among the smallest and simplest of biological systems. The investigation of their structures represented the first step toward the establishment of molecular biophysics, over half a century ago. Many research groups are now pursuing investigations of viral assembly, a process that could offer new opportunities for the design of antiviral drugs and novel nanoparticles. A variety of experimental, theoretical and computational methods have been brought to bear on the study of virus structure and assembly. In this Perspective we review the contributions of theoretical and computational approaches to our understanding of the structure, energetics, thermodynamics and assembly of DNA bacteriophage and single-stranded icosahedral RNA viruses.