Interplay between Alba and Cren7 Regulates Chromatin Compaction in Sulfolobus solfataricus.

Chromatin compaction and regulation are essential processes for the normal function of all organisms, yet knowledge on how archaeal chromosomes are packed into higher-order structures inside the cell remains elusive. In this study, we investigated the role of archaeal architectural proteins Alba and Cren7 in chromatin folding and dynamics. Atomic force microscopy revealed that Sulfolobus solfataricus chromatin is composed of 28 nm fibers and 60 nm globular structures. In vitro reconstitution showed that Alba can mediate the formation of folded DNA structures in a concentration-dependent manner. Notably, it was demonstrated that Alba on its own can form higher-order structures with DNA. Meanwhile, Cren7 was observed to affect the formation of Alba-mediated higher-order chromatin structures. Overall, the results suggest an interplay between Alba and Cren7 in regulating chromatin compaction in archaea.

Structure and Function of Archaeal Translation Initiation Factor 2 Fragments Containing Cys2-Cys2 Motifs.

The heterotrimeric (αßγ) translation initiation factor 2 of archaea and eukaryotes (a/eIF2) supplies the P-site of the ribosome with the initiation tRNA. Its two subunits (ß and γ) contain the Cys2-Cys2 motif, which is capable of forming a stable zinc finger structure in the presence of zinc ions. In this work, comparative analysis of the fragments containing Cys2-Cys2 motifs in the aIF2ß and aIF2γ structures from different organisms was carried out and their environments in crystals was analyzed. Based on the obtained data, a conclusion was made that the conformation and role of these fragments in the ß- and γ-subunits of the aIF2 are different.

Significant Loop Motions in the SsoPTP Protein Tyrosine Phosphatase Allow for Dual General Acid Functionality.

Conformational dynamics are important factors in the function of enzymes, including protein tyrosine phosphatases (PTPs). Crystal structures of PTPs first revealed the motion of a protein loop bearing a conserved catalytic aspartic acid, and subsequent nuclear magnetic resonance and computational analyses have shown the presence of motions, involved in catalysis and allostery, within and beyond the active site. The tyrosine phosphatase from the thermophilic and acidophilic Sulfolobus solfataricus (SsoPTP) displays motions of its acid loop together with dynamics of its phosphoryl-binding P-loop and the Q-loop, the first instance of such motions in a PTP. All three loops share the same exchange rate, implying their motions are coupled. Further evidence of conformational flexibility comes from mutagenesis, kinetics, and isotope effect data showing that E40 can function as an alternate general acid to protonate the leaving group when the conserved acid, D69, is mutated to asparagine. SsoPTP is not the first PTP to exhibit an alternate general acid (after VHZ and TkPTP), but E40 does not correspond to the sequence or structural location of the alternate general acids in those precedents. A high-resolution X-ray structure with the transition state analogue vanadate clarifies the role of the active site arginine R102, which varied in structures of substrates bound to a catalytically inactive mutant. The coordinated motions of all three functional loops in SsoPTP, together with the function of an alternate general acid, suggest that catalytically competent conformations are present in solution that have not yet been observed in crystal structures.

Structure of a dimer of the Sulfolobus solfataricus MCM N-terminal domain reveals a potential role in MCM ring opening.

Cells strongly regulate DNA replication to ensure genomic stability and prevent several diseases, including cancers. Eukaryotes and archaea strictly control DNA-replication initiation by the regulated loading of hexameric minichromosome maintenance (MCM) rings to encircle both strands of the DNA double helix followed by regulated activation of the loaded rings such that they then encircle one DNA strand while excluding the other. Both steps involve an open/closed ring transformation, allowing DNA strands to enter or exit. Here, the crystal structure of a dimer of the N-terminal domain of Sulfolobus solfataricus MCM with an intersubunit interface that is more extensive than in closed-ring structures, while including common interactions to enable facile interconversion, is presented. It is shown that the identified interface could stabilize open MCM rings by compensating for lost interactions at an open neighbor interface and that the prior open-ring cryo-EM structure of MCM loading has a similar extended interface adjacent to its open interface.

Insights into the mismatch discrimination mechanism of Y-family DNA polymerase Dpo4.

Nucleobases within DNA are attacked by reactive oxygen species to produce 7,8-dihydro-8-oxoguanine (oxoG) and 7,8-dihydro-8-oxoadenine (oxoA) as major oxidative lesions. The high mutagenicity of oxoG is attributed to the lesion's ability to adopt syn-oxoG:anti-dA with Watson-Crick-like geometry. Recent studies have revealed that Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) inserts nucleotide opposite oxoA in an error-prone manner and accommodates syn-oxoA:anti-dGTP with Watson-Crick-like geometry, highlighting a promutagenic nature of oxoA. To gain further insights into the bypass of oxoA by Dpo4, we have conducted kinetic and structural studies of Dpo4 extending oxoA:dT and oxoA:dG by incorporating dATP opposite templating dT. The extension past oxoA:dG was ∼5-fold less efficient than that past oxoA:dT. Structural studies revealed that Dpo4 accommodated dT:dATP base pair past anti-oxoA:dT with little structural distortion. In the Dpo4-oxoA:dG extension structure, oxoA was in an anti conformation and did not form hydrogen bonds with the primer terminus base. Unexpectedely, the dG opposite oxoA exited the primer terminus site and resided in an extrahelical site, where it engaged in minor groove contacts to the two immediate upstream bases. The extrahelical dG conformation appears to be induced by the stabilization of anti-oxoA conformation via bifurcated hydrogen bonds with Arg332. This unprecedented structure suggests that Dpo4 may use Arg332 to sense 8-oxopurines at the primer terminus site and slow the extension from the mismatch by promoting anti conformation of 8-oxopurines.

Cytochrome C with peroxidase-like activity encapsulated inside the small DPS protein nanocage.

Nature utilizes self-assembled protein-based structures as subcellular compartments in prokaryotes to sequester catalysts for specialized biochemical reactions. These protein cage structures provide unique isolated environments for the encapsulated enzymes. Understanding these systems is useful in the bioinspired design of synthetic catalytic organelle-like nanomaterials. The DNA binding protein from starved cells (Dps), isolated from Sulfolobus solfataricus, is a 9 nm dodecameric protein cage making it the smallest known naturally occurring protein cage. It is naturally over-expressed in response to oxidative stress. The small size, natural biodistribution to the kidney, and ability to cross the glomerular filtration barrier in in vivo experiments highlight its potential as a synthetic antioxidant. Cytochrome C (CytC) is a small heme protein with peroxidase-like activity involved in the electron transport chain and also plays a critical role in cellular apoptosis. Here we report the encapsulation of CytC inside the 5 nm interior cavity of Dps and demonstrate the catalytic activity of the resultant Dps nanocage with enhanced antioxidant behavior. The small cavity can accommodate a single CytC and this was achieved through self-assembly of chimeric cages comprising Dps subunits and a Dps subunit to which the CytC was fused. For selective isolation of CytC containing Dps cages, we utilized engineered polyhistidine tag present only on the enzyme fused Dps subunits (6His-Dps-CytC). The catalytic activity of encapsulated CytC was studied using guaiacol and 3,3',5,5'-tetramethylbenzidine (TMB) as two different peroxidase substrates and compared to the free (unencapsulated) CytC activity. The encapsulated CytC showed better pH dependent catalytic activity compared to free enzyme and provides a proof-of-concept model to engineer these small protein cages for their potential as catalytic nanoreactors.

Methylation deficiency of chromatin proteins is a non-mutational and epigenetic-like trait in evolved lines of the archaeon Sulfolobus solfataricus.

Archaea are a distinct and deeply rooted lineage that harbor eukaryotic-like mechanisms, including several that manage chromosome function. In previous work, the thermoacidophilic crenarchaeon, Sulfolobus solfataricus, was subjected to adaptive laboratory evolution to produce three strains, called SARC, with a new heritable trait of super acid resistance. These strains acquired heritable conserved transcriptomes, yet one strain contained no mutations. Homologous recombination without allele replacement at SARC acid resistance genes caused changes in both phenotype and expression of the targeted gene. As recombination displaces chromatin proteins, their involvement was predicted in the SARC trait. Native chromatin proteins are basic and highly abundant and undergo post-translational modification through lysine monomethylation. In this work, their modification states were investigated. In all SARC lines, two chromatin proteins, Cren7 and Sso7d, were consistently undermethylated, whereas other chromatin proteins were unaltered. This pattern was heritable in the absence of selection and independent of transient exposure to acid stress. The bulk of Sso7d was undermethylated at three contiguous N-terminal lysine residues but not at central or C-terminal regions. The N-terminal region formed a solvent-exposed patch located on the opposite side of the binding domain associated with the DNA minor groove. By analogy to eukaryotic histones, this patch could interact with other chromosomal proteins and be modulated by differential post-translational modification. Previous work established an epigenetic-like mechanism of adaptation and inheritance in S. solfataricus The identification of heritable epigenetic marks in this work further supports the occurrence of an epigenetic process in archaea.

Loop size optimization induces a strong thermal stabilization of the thioredoxin fold.

The definition of the structural basis of protein thermostability represents a major topic in structural biology and protein chemistry. We have recently observed that proteins isolated from thermophilic organisms show a better adherence to the fundamental rules of protein topology previously unveiled by Baker and coworkers (Koga et al. Nature. 2012; 491: 222-227). Here, we explored the possibility that ad hoc modifications of a natural protein following these rules could represent an efficient tool to stabilize its structure. Hence, we here designed and characterized novel variants of Escherichia coli thioredoxin (EcTrx) using a repertoire of biophysical/structural techniques. Trx chimeric variants were prepared by replacing the loop of EcTrx with the corresponding ones present in the Trxs isolated from Sulfolobus solfataricus and Sulfolobus tokodaii that show a better adherence to the topological rules. Interestingly, although the loop sequences of these proteins did not display any significant similarity, their insertion in EcTrx induced a remarkable stabilization of the protein (≥10 °C). The crystallographic structure of one of these variants corroborates the hypothesis that the optimization of the loop size is the driving force of the observed stabilization. The remarkable stabilization of the two novel chimeric Trxs, generated by applying the topological rules, represents the proof of concept that these rules may be used to stabilize natural proteins through the ad hoc optimization of the loop size. Based on the present results, we propose a novel protocol of protein stabilization that can be potentially applied to other proteins.

Global effect of the lack of inorganic polyphosphate in the extremophilic archaeon Sulfolobus solfataricus: A proteomic approach.

Inorganic polyphosphates (polyP) are present in all living cells and several important functions have been described for them. They are involved in the response to stress conditions, such as nutrient depletion, oxidative stress and toxic metals amongst others. A recombinant strain of Sulfolobus solfataricus unable to accumulate polyP was designed by the overexpression of its endogenous ppx gene. The overall impact of the lack of polyP on this S. solfataricus polyP (-) strain was analyzed by using quantitative proteomics (isotope-coded protein label, ICPL). Stress-related proteins, such as peroxiredoxins and heat shock proteins, proteins involved in metabolism and several others were produced at higher levels in the ppx expression strain. The polyP deficient strain showed an increased copper sensitivity and an earlier transcriptional up-regulation of copA gene coding for the P-type copper-exporting ATPase. This implies a complementary function of both copper resistance systems. These results strongly suggests that the lack of polyP makes this hyperthermophilic archaeon more sensitive to toxic conditions, such as an exposure to metals or other harmful stimuli, emphasizing the importance of this inorganic phosphate polymers in the adaptations to live in the environmental conditions in which thermoacidophilic archaea thrive. SIGNIFICANCE: Inorganic polyphosphate (polyP) are ubiquitous molecules with many functions in living organisms. Few studies related to these polymers have been made in archaea. The construction of a polyP deficient recombinant strain of Sulfolobus solfataricus allowed the study of the global changes in the proteome of this thermoacidophilic archaeon in the absence of polyP compared with the wild type strain. The results obtained using quantitative proteomics suggest an important participation of polyP in the oxidative stress response of the cells and as having a possible metabolic role in the cell, as previously described in bacteria. The polyP deficient strain also showed an increased copper sensitivity and an earlier transcriptional up-regulation of copA, implying a complementary role of both copper resistance systems.

Evolutionary Morphing of Tryptophan Synthase: Functional Mechanisms for the Enzymatic Channeling of Indole.

Tryptophan synthase (TrpS) is a heterotetrameric αßßα enzyme that exhibits complex substrate channeling and allosteric mechanisms and is a model system in enzymology. In this work, we characterize proposed early and late evolutionary states of TrpS and show that they have distinct quaternary structures caused by insertions-deletions of sequence segments (indels) in the ß-subunit. Remarkably, indole hydrophobic channels that connect α and ß active sites have re-emerged in both TrpS types, yet they follow different paths through the ß-subunit fold. Also, both TrpS geometries activate the α-subunit through the rearrangement of loops flanking the active site. Our results link evolutionary sequence changes in the enzyme subunits with channeling and allostery in the TrpS enzymes. The findings demonstrate that indels allow protein quaternary architectures to escape "minima" in the evolutionary landscape, thereby overcoming the conservational constraints imposed by existing functional interfaces and being free to morph into new mechanistic enzymes.

g-Tensor Directions in the Protein Structural Frame of Hyperthermophilic Archaeal Reduced Rieske-Type Ferredoxin Explored by 13C Pulsed Electron Paramagnetic Resonance.

Interpretation of magnetic resonance data in the context of structural and chemical biology requires prior knowledge of the g-tensor directions for paramagnetic metallo-cofactors with respect to the protein structural frame. Access to this information is often limited by the strict requirement of suitable protein crystals for single-crystal electron paramagnetic resonance (EPR) measurements or the reliance on protons (with ambiguous locations in crystal structures) near the paramagnetic metal site. Here we develop a novel pulsed EPR approach with selective 13Cß-cysteine labeling of model [2Fe-2S] proteins to help bypass these problems. Analysis of the 13Cß-cysteine hyperfine tensors reproduces the g-tensor of the Pseudomonas putida ISC-like [2Fe-2S] ferredoxin (FdxB). Its application to the hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin (ARF) from Sulfolobus solfataricus, for which the single-crystal EPR approach was not feasible, supports the best-fit g x-, g z-, and g y-tensor directions of the reduced cluster as nearly along Fe-Fe, S-S, and the cluster plane normal, respectively. These approximate principal directions of the reduced ARF g-tensor, explored by 13C pulsed EPR, are less skewed from the cluster molecular axes and are largely consistent with those previously determined by single-crystal EPR for the cytochrome bc1-associated, reduced Rieske [2Fe-2S] center. This suggests the approximate g-tensor directions are conserved across the phylogenetically and functionally divergent Rieske-type [2Fe-2S] proteins.

Phosphotriesterase-Magnetic Nanoparticle Bioconjugates with Improved Enzyme Activity in a Biocatalytic Membrane Reactor.

The need to find alternative bioremediation solutions for organophosphate degradation pushed the research to develop technologies based on organophosphate degrading enzymes, such as phosphotriesterase. The use of free phosphotriesterase poses limits in terms of enzyme reuse, stability, and process development. The heterogenization of enzyme on a support and their use in bioreactors implemented by membranes seems a suitable strategy, thanks to the ability of membranes to compartmentalize, to govern mass transfer, and to provide a microenvironment with tuned physicochemical and structural properties. Usually, hydrophilic membranes are used since they easily guarantee the presence of water molecules needed for the enzyme catalytic activity. However, hydrophobic materials exhibit a larger shelf life and are preferred for the construction of filters and masks. Therefore, in this work, hydrophobic polyvinylidene fluoride (PVDF) porous membranes were used to develop biocatalytic membrane reactors (BMR). The phosphotriesterase-like lactonase (PLL) enzyme ( SsoPox triple mutant from S. solfataricus) endowed with thermostable phosphotriesterase activity was used as model biocatalyst. The enzyme was covalently bound directly to the PVDF hydrophobic membrane or it was bound to magnetic nanoparticles and then positioned on the hydrophobic membrane surface by means of an external magnetic field. Investigation of kinetic properties of the two BMRs and the influence of immobilized enzyme amount revealed that the performance of the BMR was mostly dependent on the amount of enzyme and its distribution on the immobilization support. Magnetic nanocomposite mediated immobilization showed a much better performance, with an observed specific activity higher than 90% compared to grafting of the enzyme on the membrane. Even though the present work focused on phosphotriesterase, it can be easily translated to other classes of enzymes and related applications.

Crystal structure of a thermophilic O6-alkylguanine-DNA alkyltransferase-derived self-labeling protein-tag in covalent complex with a fluorescent probe.

The self-labeling protein tags are robust and versatile tools for studying different molecular aspects of cell biology. In order to be suitable for a wide spectrum of experimental conditions, it is mandatory that these systems are stable after the fluorescent labeling reaction and do not alter the properties of the fusion partner. SsOGT-H5 is an engineered variant alkylguanine-DNA-alkyl-transferase (OGT) of the hyperthermophilic archaeon Sulfolobus solfataricus, and it represents an alternative solution to the SNAP-tag® technology under harsh reaction conditions. Here we present the crystal structure of SsOGT-H5 in complex with the fluorescent probe SNAP-Vista Green® (SsOGT-H5-SVG) that reveals the conformation adopted by the protein upon the trans-alkylation reaction with the substrate, which is observed covalently bound to the catalytic cysteine residue. Moreover, we identify the amino acids that contribute to both the overall protein stability in the post-reaction state and the coordination of the fluorescent moiety stretching-out from the protein active site. We gained new insights in the conformational changes possibly occurring to the OGT proteins upon reaction with modified guanine base bearing bulky adducts; indeed, our structural analysis reveals an unprecedented conformation of the active site loop that is likely to trigger protein destabilization and consequent degradation. Interestingly, the SVG moiety plays a key role in restoring the interaction between the N- and C-terminal domains of the protein that is lost following the new conformation adopted by the active site loop in the SsOGT-H5-SVG structure. Molecular dynamics simulations provide further information into the dynamics of SsOGT-H5-SVG structure, highlighting the role of the fluorescent ligand in keeping the protein stable after the trans-alkylation reaction.

Conformational Flexibility of the Benzyl-Guanine Adduct in a Bypass Polymerase Active Site Permits Replication: Insights from Molecular Dynamics Simulations.

Although translesion synthesis (TLS) polymerases play key roles in replicating DNA that contains nucleobase addition products (adducts), there are many unknowns about their function. The present work gains indispensable structural insights from molecular dynamics simulations on the replication of O6-benzyl-guanine (Bz-dG) prior to bond formation during dCTP insertion opposite the adduct by Dpo4. When combined with previous X-ray crystal structures of the Bz-dG extension complex, molecular details are now available for each stage during a single TLS replication cycle for this carcinogenic lesion. Our calculations illustrate that Bz-dG preferentially adopts an intercalated bulky moiety orientation in the Dpo4 preinsertion complex, which stabilizes the complex through Bz-dG interactions with the previously replicated 3'-base pair and positions the carcinogenic group in the dNTP binding site. Nevertheless, the maintained inherent flexibility of Bz-dG due to a stark lack of interactions with the polymerase or template DNA allows the bulky moiety to adopt a major groove position opposite an incoming dCTP in an orientation that is conducive for the experimentally observed nonmutagenic bypass. Comparison of Bz-dG and canonical dG replication clarifies that the experimentally observed decrease in dCTP binding affinity and replication efficiency upon adduct formation is likely caused by a combination of factors, including the required template nucleotide conformational change and destabilized template-dCTP hydrogen bonding. Although additional aspects of the replication process, such as the impact of the adduct on the nucleotidyl-transfer reaction, may also be important for fully rationalizing experimental replication data and must be considered in future work, the present contribution emphasizes the importance of considering the effect of DNA damage on the early stages of the TLS process.

Increased Processivity, Misincorporation, and Nucleotide Incorporation Efficiency in Sulfolobus solfataricus Dpo4 Thumb Domain Mutants.

The present study aimed to increase the processivity of Sulfolobus solfataricus DNA polymerase Dpo4. Protein engineering and bioinformatics were used to compile a library of potential Dpo4 mutation sites. Ten potential mutants were identified and constructed. A primer extension assay was used to evaluate the processivity of Dpo4 mutants. Thumb (A181D) and finger (E63K) domain mutants showed a processivity of 20 and 19 nucleotides (nt), respectively. A little finger domain mutant (I248Y) exhibited a processivity of 17 nt, only 1 nt more than wild-type Dpo4. Furthermore, the A181D mutant showed lower fidelity and higher nucleotide incorporation efficiency (4.74 × 10-4 s-1 µM-1) than E63K and I248Y mutants. When tasked with bypassing damage, the A181D mutant exhibited a 3.81-fold and 2.62-fold higher catalytic efficiency (kcat/Km ) at incorporating dCTP and dATP, respectively, than wild-type Dpo4. It also showed a 55% and 91.5% higher catalytic efficiency when moving beyond the damaged 8-oxoG:C and 8-oxoG:A base pairs, respectively, compared to wild-type Dpo4. Protein engineering and bioinformatics methods can effectively increase the processivity and translesion synthesis ability of Dpo4.IMPORTANCE DNA polymerases with poor fidelity can be exploited to store data and record changes in response to the intracellular environment. Sulfolobus solfataricus Dpo4 is such an enzyme, although its use is hindered by its low processivity. In this work, we used a bioinformatics and protein engineering approach to generate Dpo4 mutants with improved processivity. We identified the Dpo4 thumb domain as the most relevant in controlling processivity.

DNA binding strength increases the processivity and activity of a Y-Family DNA polymerase.

DNA polymerase (pol) processivity, i.e., the bases a polymerase extends before falling off the DNA, and activity are important for copying difficult DNA sequences, including simple repeats. Y-family pols would be appealing for copying difficult DNA and incorporating non-natural dNTPs, due to their low fidelity and loose active site, but are limited by poor processivity and activity. In this study, the binding between Dbh and DNA was investigated to better understand how to rationally design enhanced processivity in a Y-family pol. Guided by structural simulation, a fused pol Sdbh with non-specific dsDNA binding protein Sso7d in the N-terminus was designed. This modification increased in vitro processivity 4-fold as compared to the wild-type Dbh. Additionally, bioinformatics was used to identify amino acid mutations that would increase stabilization of Dbh bound to DNA. The variant SdbhM76I further improved the processivity of Dbh by 10 fold. The variant SdbhKSKIP241-245RVRKS showed higher activity than Dbh on the incorporation of dCTP (correct) and dATP (incorrect) opposite the G (normal) or 8-oxoG(damaged) template base. These results demonstrate the capability to rationally design increases in pol processivity and catalytic efficiency through computational DNA binding predictions and the addition of non-specific DNA binding domains.

Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme.

Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.

Ion- and water-binding sites inside an occluded hourglass pore of a trimeric intracellular cation (TRIC) channel.

BACKGROUND: Trimeric intracellular cation (TRIC) channels are crucial for Ca2+ handling in eukaryotes and are involved in K+ uptake in prokaryotes. Recent studies on the representative members of eukaryotic and prokaryotic TRIC channels demonstrated that they form homotrimeric units with the ion-conducting pores contained within each individual monomer. RESULTS: Here we report detailed insights into the ion- and water-binding sites inside the pore of a TRIC channel from Sulfolobus solfataricus (SsTRIC). Like the mammalian TRIC channels, SsTRIC is permeable to both K+ and Na+ with a slight preference for K+, and is nearly impermeable to Ca2+, Mg2+, or Cl-. In the 2.2-Å resolution K+-bound structure of SsTRIC, ion/water densities have been well resolved inside the pore. At the central region, a filter-like structure is shaped by the kinks on the second and fifth transmembrane helices and two nearby phenylalanine residues. Below the filter, the cytoplasmic vestibule is occluded by a plug-like motif attached to an array of pore-lining charged residues. CONCLUSIONS: The asymmetric filter-like structure at the pore center of SsTRIC might serve as the basis for the channel to bind and select monovalent cations. A Velcro-like plug-pore interacting model has been proposed and suggests a unified framework accounting for the gating mechanisms of prokaryotic and eukaryotic TRIC channels.

The Rrp4-exosome complex recruits and channels substrate RNA by a unique mechanism.

The exosome is a large molecular machine involved in RNA degradation and processing. Here we address how the trimeric Rrp4 cap enhances the activity of the archaeal enzyme complex. Using methyl-TROSY NMR methods we identified a 50-Å long RNA binding path on each Rrp4 protomer. We show that the Rrp4 cap can thus simultaneously recruit three substrates, one of which is degraded in the core while the others are positioned for subsequent degradation rounds. The local interaction energy between the substrate and the Rrp4-exosome increases from the periphery of the complex toward the active sites. Notably, the intrinsic interaction strength between the cap and the substrate is weakened as soon as substrates enter the catalytic barrel, which provides a means to reduce friction during substrate movements toward the active sites. Our data thus reveal a sophisticated exosome-substrate interaction mechanism that enables efficient RNA degradation.

Correlation of fitness landscapes from three orthologous TIM barrels originates from sequence and structure constraints.

Sequence divergence of orthologous proteins enables adaptation to environmental stresses and promotes evolution of novel functions. Limits on evolution imposed by constraints on sequence and structure were explored using a model TIM barrel protein, indole-3-glycerol phosphate synthase (IGPS). Fitness effects of point mutations in three phylogenetically divergent IGPS proteins during adaptation to temperature stress were probed by auxotrophic complementation of yeast with prokaryotic, thermophilic IGPS. Analysis of beneficial mutations pointed to an unexpected, long-range allosteric pathway towards the active site of the protein. Significant correlations between the fitness landscapes of distant orthologues implicate both sequence and structure as primary forces in defining the TIM barrel fitness landscape and suggest that fitness landscapes can be translocated in sequence space. Exploration of fitness landscapes in the context of a protein fold provides a strategy for elucidating the sequence-structure-fitness relationships in other common motifs.