Profiling of lipids in Thermus thermophilus HB8 grown under various conditions.

The membrane lipids of Thermus species have unique structures. Only four polar lipid species have so far been identified in Thermus thermophilus HB8; namely, are two phosphoglycolipids and two glycolipids, both of which have three branched fatty acid chains. Other lipid molecules may be present; however, they have not been identified so far. To clarify the whole lipid profile of T. thermophilus HB8, we cultured this organism under four different growth (temperature and/or nutrition) conditions and analyzed the compositions of polar lipids and fatty acids by high-performance thin-layer chromatography (HPTLC) and gas chromatograph-mass spectrometry (GCï½°MS), respectively. Thirty-one lipid spots were detected on HPTLC plates and profiled in terms of the presence or absence of phosphate, amino, and sugar groups. Then, we allocated ID numbers to all the spots. Comparative analyses of these polar lipids showed that the diversity of lipid molecules increased under high temperature and minimal medium conditions. In particular, aminolipid species increased under high temperature conditions. As for the fatty acid comparison by GC-MS, iso-branched even-numbered carbon atoms, which are unusual in this organism, significantly increased under the minimal medium condition, suggesting that kinds of branched amino acids at the fatty acid terminus varies under different nutrition conditions. In this study, several unidentified lipids were detected, and elucidation of the lipid structures will provide important information on the environmental adaptation of bacteria.

Structure of the hypothetical protein TTHA1873 from Thermus thermophilus.

The crystal structure of an uncharacterized hypothetical protein, TTHA1873 from Thermus thermophilus, has been determined by X-ray crystallography to a resolution of 1.78 Šusing the single-wavelength anomalous dispersion method. The protein crystallized as a dimer in two space groups: P43212 and P6122. Structural analysis of the hypothetical protein revealed that the overall fold of TTHA1873 has a ß-sandwich jelly-roll topology with nine ß-strands. TTHA1873 is a dimeric metal-binding protein that binds to two Ca2+ ions per chain, with one on the surface and the other stabilizing the dimeric interface of the two chains. A structural homology search indicates that the protein has moderate structural similarity to one domain of cell-surface proteins or agglutinin receptor proteins. Red blood cells showed visible agglutination at high concentrations of the hypothetical protein.

Crystal structure of thermally stable homodimeric cytochrome c'-ß from Thermus thermophilus.

Cytochrome c'-ß is a heme protein that belongs to the cytochrome P460 family and consists of homodimeric subunits with a predominantly antiparallel ß-sheet fold. Here, the crystal structure of cytochrome c'-ß from the thermophilic Thermus thermophilus (TTCP-ß) is reported at 1.74 Šresolution. TTCP-ß has a typical antiparallel ß-sheet fold similar to that of cytochrome c'-ß from the moderately thermophilic Methylococcus capsulatus (MCCP-ß). The phenylalanine cap structure around the distal side of the heme is also similar in TTCP-ß and MCCP-ß, indicating that both proteins similarly bind nitric oxide and carbon monoxide, as observed spectroscopically. Notably, TTCP-ß exhibits a denaturation temperature of 117°C, which is higher than that of MCCP-ß. Mutational analysis reveals that the increased homodimeric interface area of TTCP-ß contributes to its high thermal stability. Furthermore, 14 proline residues, which are mostly located in the TTCP-ß loop regions, possibly contribute to the rigid loop structure compared with MCCP-ß, which has only six proline residues. These findings, together with those from phylogenetic analysis, suggest that the structures of Thermus cytochromes c'-ß, including TTCP-ß, are optimized for function under the high-temperature conditions in which the source organisms live.

Electrochemical characterization of an engineered red copper protein featuring an unprecedented entropic control of the reduction potential.

Copper is a ubiquitous metal in biology that, among other functions, is implicated in enzymatic redox catalysis and in protein electron transfer (ET). When it comes to ET, copper sites are found in two main forms, mononuclear type 1 (T1) and binuclear CuA sites, which share a common cupredoxin fold. Other relevant copper sites are the so-called type 2 (T2), which are more resilient to undergo direct electrochemistry and are usually involved in catalysis. Here we report the electrochemical and spectroscopic characterization of a novel T2-like copper site engineered following the loop swapping strategy. The ligand loop sequence of the newly discovered T1 copper site from Nitrosopumilus maritimus was introduced into the CuA scaffold from Thermus thermophilus yielding a chimeric protein that shows spectroscopic features different from both parental proteins, and resemble those of red T2 copper sites, albeit with a shorter Cu-S(Cys) bond length. The novel T2 site undergoes efficient direct electrochemistry, which allows performing temperature-dependent cyclic voltammetry studies. The obtained results reveal that this chimera constitutes the first example of a copper protein with entropically controlled reduction potential, thereby contrasting the enthalpic supremacy observed for all other copper sites reported so far. The underlying bases for this entropic control are critically discussed.

A Hyperthermoactive-Cas9 Editing Tool Reveals the Role of a Unique Arsenite Methyltransferase in the Arsenic Resistance System of Thermus thermophilus HB27.

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to humans. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pulldown assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosyl-l-methionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyze SAM-dependent arsenite methylation with formation of monomethylarsenites (MMAs) and dimethylarsenites (DMAs). In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9-based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus ΔTtarsM genome was substituted by a gene encoding a stabilized yellow fluorescent protein (sYFP) to create a sensitive genome-based bioreporter system for the detection of arsenic ions. IMPORTANCE We here describe the discovery of an unknown protein by using a proteomics approach with a transcriptional regulator as bait. Remarkably, we successfully obtained a novel type of enzyme through the interaction with a transcriptional regulator controlling the expression of this enzyme. Employing this strategy, we isolated TtArsM, the first thermophilic prokaryotic arsenite methyltransferase, as a new enzyme of the arsenic resistance mechanism in T. thermophilus HB27. The atypical arsenite binding site of TtArsM categorizes the enzyme as the first member of a new arsenite methyltransferase type, exclusively present in the Thermus genus. The enzyme methylates arsenite-producing MMAs and DMAs. Furthermore, we developed an hyperthermophilic Cas9-based genome-editing tool, active up to 65°C. The tool allowed us to perform highly efficient, marker-free modifications (either gene deletion or insertion) in the T. thermophilus genome. With these modifications, we confirmed the critical role of TtArsM in the arsenite detoxification system and developed a sensitive whole-cell bioreporter for arsenic ions. We anticipate that the developed tool can be easily adapted for editing the genomes of other thermophilic bacteria, significantly boosting fundamental and metabolic engineering in hyperthermophilic microorganisms.

Structural determinants of a permeation barrier of the SecYEG translocon in the active state.

The SecYEG translocon is a channel in bacteria, which provides a passage for secretory proteins across as well as integration of membrane proteins into the plasma membrane. The molecular mechanism, by which SecYEG manages protein transport while preventing water and ion leakage through the membrane, is still controversial. We employed molecular dynamics simulations to assess the contribution of the major structural elements - the plug and the pore ring (PR) - to the sealing of SecYEG in the active state, i.e., with a signal sequence helix occupying the lateral gate. We found, that the PR alone can provide a very tight seal for the wild-type translocon in the active state for both water and ions. Simulations of the mutant I403N, in which one of the PR-defining isoleucine residues is replaced with asparagine, suggest that hydrophobic interactions within the PR and between the PR and the plug are important for maintaining a tight conformation of the wild-type channel around the PR. Disruption of these interactions results in strong fluctuations of helix TM7 and water leakage of the translocon.

Coupled transport of electrons and protons in a bacterial cytochrome c oxidase-DFT calculated properties compared to structures and spectroscopies.

After a general introduction to the features and mechanisms of cytochrome c oxidases (CcOs) in mitochondria and aerobic bacteria, we present DFT calculated physical and spectroscopic properties for the catalytic reaction cycle compared with experimental observations in bacterial ba3 type CcO, also with comparisons/contrasts to aa3 type CcOs. The Dinuclear Complex (DNC) is the active catalytic reaction center, containing a heme a3 Fe center and a near lying Cu center (called CuB) where by successive reduction and protonation, molecular O2 is transformed to two H2O molecules, and protons are pumped from an inner region across the membrane to an outer region by transit through the CcO integral membrane protein. Structures, energies and vibrational frequencies for Fe-O and O-O modes are calculated by DFT over the catalytic cycle. The calculated DFT frequencies in the DNC of CcO are compared with measured frequencies from Resonance Raman spectroscopy to clarify the composition, geometry, and electronic structures of different intermediates through the reaction cycle, and to trace reaction pathways. X-ray structures of the resting oxidized state are analyzed with reference to the known experimental reaction chemistry and using DFT calculated structures in fitting observed electron density maps. Our calculations lead to a new proposed reaction pathway for coupling the PR → F → OH (ferryl-oxo → ferric-hydroxo) pathway to proton pumping by a water shift mechanism. Through this arc of the catalytic cycle, major shifts in pKa's of the special tyrosine and a histidine near the upper water pool activate proton transfer. Additional mechanisms for proton pumping are explored, and the role of the CuB+ (cuprous state) in controlling access to the dinuclear reaction site is proposed.

Amyloidogenic Propensities of Ribosomal S1 Proteins: Bioinformatics Screening and Experimental Checking.

Structural S1 domains belong to the superfamily of oligosaccharide/oligonucleotide-binding fold domains, which are highly conserved from prokaryotes to higher eukaryotes and able to function in RNA binding. An important feature of this family is the presence of several copies of the structural domain, the number of which is determined in a strictly limited range from one to six. Despite the strong tendency for the aggregation of several amyloidogenic regions in the family of the ribosomal S1 proteins, their fibril formation process is still poorly understood. Here, we combined computational and experimental approaches for studying some features of the amyloidogenic regions in this protein family. The FoldAmyloid, Waltz, PASTA 2.0 and Aggrescan programs were used to assess the amyloidogenic propensities in the ribosomal S1 proteins and to identify such regions in various structural domains. The thioflavin T fluorescence assay and electron microscopy were used to check the chosen amyloidogenic peptides' ability to form fibrils. The bioinformatics tools were used to study the amyloidogenic propensities in 1331 ribosomal S1 proteins. We found that amyloidogenicity decreases with increasing sizes of proteins. Inside one domain, the amyloidogenicity is higher in the terminal parts. We selected and synthesized 11 amyloidogenic peptides from the Escherichia coli and Thermus thermophilus ribosomal S1 proteins and checked their ability to form amyloids using the thioflavin T fluorescence assay and electron microscopy. All 11 amyloidogenic peptides form amyloid-like fibrils. The described specific amyloidogenic regions are actually responsible for the fibrillogenesis process and may be potential targets for modulating the amyloid properties of bacterial ribosomal S1 proteins.

Mechanical inhibition of isolated Vo from V/A-ATPase for proton conductance.

V-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis in the hydrophilic V1 domain, with proton flow through the Vo membrane domain, via rotation of the central rotor complex relative to the surrounding stator apparatus. Upon dissociation from the V1 domain, the Vo domain of the eukaryotic V-ATPase can adopt a physiologically relevant auto-inhibited form in which proton conductance through the Vo domain is prevented, however the molecular mechanism of this inhibition is not fully understood. Using cryo-electron microscopy, we determined the structure of both the holo V/A-ATPase and isolated Vo at near-atomic resolution, respectively. These structures clarify how the isolated Vo domain adopts the auto-inhibited form and how the holo complex prevents formation of the inhibited Vo form.

Common mechanism of thermostability in small α- and ß-proteins studied by molecular dynamics.

Protein thermostability is important to evolution, diseases, and industrial applications. Proteins use diverse molecular strategies to achieve stability at high temperature, yet reducing the entropy of unfolding seems required. We investigated five small α-proteins and five ß-proteins with known, distinct structures and thermostability (Tm ) using multi-seed molecular dynamics simulations at 300, 350, and 400 K. The proteins displayed diverse changes in hydrogen bonding, solvent exposure, and secondary structure with no simple relationship to Tm . Our dynamics were in good agreement with experimental B-factors at 300 K and insensitive to force-field choice. Despite the very distinct structures, the native-state (300 + 350 K) free-energy landscapes (FELs) were significantly broader for the two most thermostable proteins and smallest for the three least stable proteins in both the α- and ß-group and with both force fields studied independently (tailed t-test, 95% confidence level). Our results suggest that entropic ensembles stabilize proteins at high temperature due to reduced entropy of unfolding, viz., ΔG = ΔH - TΔS. Supporting this mechanism, the most thermostable proteins were also the least kinetically stable, consistent with broader FELs, typified by villin headpiece and confirmed by specific comparison to a mesophilic ortholog of Thermus thermophilus apo-pyrophosphate phosphohydrolase. We propose that molecular strategies of protein thermostabilization, although diverse, tend to converge toward highest possible entropy in the native state consistent with the functional requirements. We speculate that this tendency may explain why many proteins are not optimally structured and why molten-globule states resemble native proteins so much.

Crystal Structure of α-Galactosidase from Thermus thermophilus: Insight into Hexamer Assembly and Substrate Specificity.

α-Galactosidase catalyzes the hydrolysis of a terminal α-galactose residue in galacto-oligosaccharides and has potential in various industrial applications and food processing. We determined the crystal structures of α-galactosidase from the thermophilic microorganism Thermus thermophilus (TtGalA) and its complexes with pNPGal and stachyose. The monomer folds into an N-terminal domain, a catalytic (ß/α)8 barrel domain, and a C-terminal domain. The domain organization is similar to that of the other family of 36 α-galactosidases, but TtGalA presents a cagelike hexamer. Structural analysis shows that oligomerization may be a key factor for the thermal adaption of TtGalA. The structure of TtGalA complexed with stachyose reveals only the existence of one -1 subsite and one +1 subsite in the active site. Structural comparison of the stachyose-bound complexes of TtGalA and GsAgaA, a tetrameric enzyme with four subsites, suggests evolutionary divergence of substrate specificity within the GH36 family of α-galactosidases. To the best of our knowledge, the crystal structure of TtGalA is the first report of a quaternary structure as a hexameric assembly in the α-galactosidase family.

Cryo-electron microscopy reveals two distinct type IV pili assembled by the same bacterium.

Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus ('wide' and 'narrow'), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility.

A fully orthogonal system for protein synthesis in bacterial cells.

Ribosome engineering is a powerful approach for expanding the catalytic potential of the protein synthesis apparatus. Due to the potential detriment the properties of the engineered ribosome may have on the cell, the designer ribosome needs to be functionally isolated from the translation machinery synthesizing cellular proteins. One solution to this problem was offered by Ribo-T, an engineered ribosome with tethered subunits which, while producing a desired protein, could be excluded from general translation. Here, we provide a conceptually different design of a cell with two orthogonal protein synthesis systems, where Ribo-T produces the proteome, while the dissociable ribosome is committed to the translation of a specific mRNA. The utility of this system is illustrated by generating a comprehensive collection of mutants with alterations at every rRNA nucleotide of the peptidyl transferase center and isolating gain-of-function variants that enable the ribosome to overcome the translation termination blockage imposed by an arrest peptide.

Charge Transfer and Chemo-Mechanical Coupling in Respiratory Complex I.

The mitochondrial respiratory chain, formed by five protein complexes, utilizes energy from catabolic processes to synthesize ATP. Complex I, the first and the largest protein complex of the chain, harvests electrons from NADH to reduce quinone, while pumping protons across the mitochondrial membrane. Detailed knowledge of the working principle of such coupled charge-transfer processes remains, however, fragmentary due to bottlenecks in understanding redox-driven conformational transitions and their interplay with the hydrated proton pathways. Complex I from Thermus thermophilus encases 16 subunits with nine iron-sulfur clusters, reduced by electrons from NADH. Here, employing the latest crystal structure of T. thermophilus complex I, we have used microsecond-scale molecular dynamics simulations to study the chemo-mechanical coupling between redox changes of the iron-sulfur clusters and conformational transitions across complex I. First, we identify the redox switches within complex I, which allosterically couple the dynamics of the quinone binding pocket to the site of NADH reduction. Second, our free-energy calculations reveal that the affinity of the quinone, specifically menaquinone, for the binding-site is higher than that of its reduced, menaquinol form-a design essential for menaquinol release. Remarkably, the barriers to diffusive menaquinone dynamics are lesser than that of the more ubiquitous ubiquinone, and the naphthoquinone headgroup of the former furnishes stronger binding interactions with the pocket, favoring menaquinone for charge transport in T. thermophilus. Our computations are consistent with experimentally validated mutations and hierarchize the key residues into three functional classes, identifying new mutation targets. Third, long-range hydrogen-bond networks connecting the quinone-binding site to the transmembrane subunits are found to be responsible for proton pumping. Put together, the simulations reveal the molecular design principles linking redox reactions to quinone turnover to proton translocation in complex I.

Cryo-EM structure in situ reveals a molecular switch that safeguards virus against genome loss.

The portal protein is a key component of many double-stranded DNA viruses, governing capsid assembly and genome packaging. Twelve subunits of the portal protein define a tunnel, through which DNA is translocated into the capsid. It is unknown how the portal protein functions as a gatekeeper, preventing DNA slippage, whilst allowing its passage into the capsid, and how these processes are controlled. A cryo-EM structure of the portal protein of thermostable virus P23-45, determined in situ in its procapsid-bound state, indicates a mechanism that naturally safeguards the virus against genome loss. This occurs via an inversion of the conformation of the loops that define the constriction in the central tunnel, accompanied by a hydrophilic-hydrophobic switch. The structure also shows how translocation of DNA into the capsid could be modulated by a changing mode of protein-protein interactions between portal and capsid, across a symmetry-mismatched interface.

Application of solid-phase DNA probe method with cleavage by deoxyribozyme for analysis of long non-coding RNAs.

The solid-phase DNA probe method is a well-established technique for tRNA purification. We have applied this method for purification and analysis of other non-coding RNAs. Three columns for purification of tRNAPhe, transfer-messenger RNA (tmRNA) and 16S rRNA from Thermus thermophilus were connected in tandem and purifications were performed. From each column, tRNAPhe, tmRNA and 16S rRNA could be purified in a single step. This is the first report of purification of native tmRNA from T. thermophilus and the purification demonstrates that the solid-phase DNA probe method is applicable to non-coding RNA, which is present in lower amounts than tRNA. Furthermore, if a long non-coding RNA is cleaved site-specifically and the fragment can be purified by the solid-phase DNA probe method, modified nucleosides in the long non-coding RNA can be analysed. Therefore, we designed a deoxyribozyme (DNAzyme) to perform site-specific cleavage of 16S rRNA, examined optimum conditions and purified the resulting RNA fragment. Sequencing of complimentary DNA and mass spectrometric analysis revealed that the purified RNA corresponded to the targeted fragment of 16S rRNA. Thus, the combination of DNAzyme cleavage and purification using solid-phase DNA probe methodology can be a useful technique for analysis of modified nucleosides in long non-coding RNAs.

Unifying the Aminohexopyranose- and Peptidyl-Nucleoside Antibiotics: Implications for Antibiotic Design.

In search of new anti-tuberculars compatible with anti-retroviral therapy we re-identified amicetin as a lead compound. Amicetin's binding to the 70S ribosomal subunit of Thermus thermophilus (Tth) has been unambiguously determined by crystallography and reveals it to occupy the peptidyl transferase center P-site of the ribosome. The amicetin binding site overlaps significantly with that of the well-known protein synthesis inhibitor balsticidin S. Amicetin, however, is the first compound structurally characterized to bind to the P-site with demonstrated selectivity for the inhibition of prokaryotic translation. The natural product-ribosome structure enabled the synthesis of simplified analogues that retained both potency and selectivity for the inhibition of prokaryotic translation.

Structural and thermodynamic correlation illuminates the selective transport mechanism of disaccharide α-glycosides through ABC transporter.

Carbohydrate (or sugar) molecules are extremely diverse regarding their length, linkage and epimeric state. Selective acquisition of these molecules inside the cell is achieved by the substrate (or solute)-binding protein of ATP-binding cassette (ABC) transport system. However, the molecular mechanism underlying the selective transport of diverse carbohydrates remains unclear mainly owing to their structural complexity and stereochemistry. This study reports crystal structures of an α-glycoside-binding protein (αGlyBP, ORF ID: TTHA0356 from Thermus thermophilus HB8) in complex with disaccharide α-glycosides namely trehalose (α-1,1), sucrose (α-1,2), maltose (α-1,4), palatinose (α-1,6) and glucose within a resolution range of 1.6-2.0 Å. Despite transporting multiple types of sugars, αGlyBP maintains its stereoselectivity for both glycosidic linkage as well as an epimeric hydroxyl group. Out of the two subsites identified in the active-site pocket, subsite B which accommodates the glucose and glycosyl unit of disaccharide α-glycosides is highly conserved. In addition, structural data confirms the paradoxical behavior of glucose, where it replaces the high-affinity ligand(s) (disaccharide α-glycosides) from the active site of the protein. Comparative assessment of open and closed conformations of αGlyBP along with mutagenic and thermodynamic studies identifies the hinge region as the first interaction site for the ligands. On the other hand, encapsulation of ligand inside the active site is achieved through the N-terminal domain (NTD) movement, whereas the C-terminal domain (CTD) of αGlyBP is identified to be rigid and postulated to be responsible for maintaining the interaction with the transmembrane domain (TMD) during substrate translocation. DATABASE: Structural data are available in RCSB Protein Data Bank under the accession number(s) 6J9W, 6J9Y, 6JAD, 6JAG, 6JAH, 6JAI, 6JAL, 6JAM, 6JAN, 6JAO, 6JAP, 6JAQ, 6JAR, 6JAZ, 6JB0, 6JB4, 6JBA, 6JBB and 6JBE.

Arabidopsis thaliana Hcc1 is a Sco-like metallochaperone for CuA assembly in Cytochrome c Oxidase.

The assembly of the CuA site in Cytochrome c Oxidase (COX) is a critical step for aerobic respiration in COX-dependent organisms. Several gene products have been associated with the assembly of this copper site, the most conserved of them belonging to the Sco family of proteins, which have been shown to perform different roles in different organisms. Plants express two orthologs of Sco proteins: Hcc1 and Hcc2. Hcc1 is known to be essential for plant development and for COX maturation, but its precise function has not been addressed until now. Here, we report the biochemical, structural and functional characterization of Arabidopsis thaliana Hcc1 protein (here renamed Sco1). We solved the crystal structure of the Cu+1 -bound soluble domain of this protein, revealing a tri coordinated environment involving a CxxxCxn H motif. We show that AtSco1 is able to work as a copper metallochaperone, inserting two Cu+1 ions into the CuA site in a model of CoxII. We also show that AtSco1 does not act as a thiol-disulfide oxido-reductase. Overall, this information sheds new light on the biochemistry of Sco proteins, highlighting the diversity of functions among them despite their high structural similarities. DATABASE: PDB entry 6N5U (Crystal structure of Arabidopsis thaliana ScoI with copper bound).

Gene Silencing Mechanisms Revealed by Dynamics of Guide, Target, and Duplex Binding to Argonaute.

Argonaute (Ago) protein plays a central role in silencing gene expression by binding a "guide" strand to the base-pair with a complementary mRNA and degrading the mRNA. The current understanding of how Ago-guide and Ago-guide-mRNA complexes assemble is based mainly on static crystal structures; the associated kinetic pathways remain unknown/unclear. By simulating the successive binding of guide/target strand to Thermus thermophilus Ago (TtAgo) and computing the respective free energy landscapes, we directly visualize how TtAgo silencing complexes form and function. We show that the guide binding rate depends on its initial loading position onto TtAgo. Subsequent target recognition beyond the scissile 10-11 nucleotides must overcome a substantial energy barrier for TtAgo's nucleotide-binding groove to expand widely. This work reveals novel roles for the core TtAgo domains and shows how the kinetic barriers that must be overcome for critical structural changes to occur lead to target repression/cleavage.