Tertiary Structural Motifs Research Articles

Optimized periphery-core interface increases fitness of the Bacillus subtilisglmS ribozyme.

Like other functional RNAs, ribozymes encode a conserved catalytic center supported by peripheral domains that vary among ribozyme sub-families. To understand how core-periphery interactions contribute to ribozyme fitness, we compared the cleavage kinetics of all single base substitutions at 152 sites across the Bacillus subtilis glmS ribozyme by high-throughput sequencing (k-seq). The in vitro activity map mirrored phylogenetic sequence conservation in glmS ribozymes, indicating that biological fitness reports all biochemically important positions. The k-seq results and folding assays showed that most deleterious mutations lower activity by impairing ribozyme self-assembly. All-atom molecular dynamics simulations of the complete ribozyme revealed how individual mutations in the core or the IL4 peripheral loop introduce a non-native tertiary interface that rewires the catalytic center, eliminating activity. We conclude that the need to avoid non-native helix packing powerfully constrains the evolution of tertiary structure motifs in RNA.

Dissecting the energetic architecture within an RNA tertiary structural motif via high-throughput thermodynamic measurements

Structured RNAs and RNA/protein complexes perform critical cellular functions. They often contain structurally conserved tertiary contact "motifs," whose occurrence simplifies the RNA folding landscape. Prior studies have focused on the conformational and energetic modularity of intact motifs. Here, we turn to the dissection of one common motif, the 11nt receptor (11ntR), using quantitative analysis of RNA on a massively parallel array to measure the binding of all single and double 11ntR mutants to GAAA and GUAA tetraloops, thereby probing the energetic architecture of the motif. While the 11ntR behaves as a motif, its cooperativity is not absolute. Instead, we uncovered a gradient from high cooperativity amongst base-paired and neighboring residues to additivity between distant residues. As expected, substitutions at residues in direct contact with the GAAA tetraloop resulted in the largest decreases to binding, and energetic penalties of mutations were substantially smaller for binding to the alternate GUAA tetraloop, which lacks tertiary contacts present with the canonical GAAA tetraloop. However, we found that the energetic consequences of base partner substitutions are not, in general, simply described by base pair type or isostericity. We also found exceptions to the previously established stability-abundance relationship for 11ntR sequence variants. These findings of "exceptions to the rule" highlight the power of systematic high-throughput approaches to uncover novel variants for future study in addition to providing an energetic map of a functional RNA.

Arabidopsis clathrin adaptor EPSIN1 but not MODIFIED TRANSPORT TO THE VACOULE1 contributes to effective plant immunity against pathogenic Pseudomonas bacteria

ABSTRACT In eukaryotes, EPSINs are Epsin N-terminal Homology (ENTH) domain-containing proteins that serve as monomeric clathrin adaptors at the plasma membrane (PM) or the trans-Golgi Network (TGN)/early endosomes (EE). The model plant Arabidopsis thaliana encodes for seven ENTH proteins, of which so far, only AtEPSIN1 (AtEPS1) and MODIFIED TRANSPORT TO THE VACUOLE1 (AtMTV1) localize to the TGN/EE and contribute to cargo trafficking to both the cell surface and the vacuole. However, relatively little is known about role(s) of any plant EPSIN in governing physiological responses. We have recently shown that AtEPS1 is a positive modulator of plant immune signaling and pattern-triggered immunity against flagellated Pseudomonas syringae pv. tomato (Pto) DC3000 bacteria. In eps1 mutants, impaired immune responses correlate with reduced accumulation of the receptor FLAGELLIN SENSING2 (AtFLS2) and the convergent immune co-receptor BRASSINOSTEROID INSENTIVE1-ASSOCIATED RECEPTOR KINASE1 (AtBAK1) in the PM. Here, we report that in contrast to AtEPS1, the TGN/EE-localized AtMTV1 did not contribute significantly to immunity against pathogenic Pto DC3000 bacteria. We also compared the amino acid sequences, peptide motif structures and in silico tertiary structures of the ENTH domains of AtEPS1 and AtMTV1 in more detail. We conclude that despite sharing the classical tertiary alpha helical ENTH-domain structure and clathrin-binding motifs, the overall low amino acid identity and differences in peptide motifs may explain their role(s) in trafficking of some of the same as well as distinct cargo components to their site of function, with the latter potentially contributing to differences in physiological responses.

G4Atlas: a comprehensive transcriptome-wide G-quadruplex database.

RNA G-quadruplex (rG4) is a vital RNA tertiary structure motif that involves the base pairs on both Hoogsteen and Watson-Crick faces of guanines. rG4 is of great importance in the post-transcriptional regulation of gene expression. Experimental technologies have advanced to identify in vitro and in vivo rG4s across diverse transcriptomes. Building on these recent advances, here we present G4Atlas, the first transcriptome-wide G-quadruplex database, in which we have collated, classified, and visualized transcriptome rG4 experimental data, generated from rG4-seq, chemical profiling and ligand-binding methods. Our comprehensive database includes transcriptome-wide rG4s generated from 82 experimental treatments and 238 samples across ten species. In addition, we have also included RNA secondary structure prediction information across both experimentally identified and unidentified rG4s to enable users to display any potential competitive folding between rG4 and RNA secondary structures. As such, G4Atlas will enable users to explore the general functions of rG4s in diverse biological processes. In addition, G4Atlas lays the foundation for further data-driven deep learning algorithms to examine rG4 structural features.

Design and Synthesis of Crosslinked Helix Dimers as Protein Tertiary Structure Mimics.

Crosslinked helix dimers (CHDs) are synthetic tertiary helical structure motifs designed to modulate interactions of proteins with binding partners. Helix dimers serve as mimics of coiled coils, which are known to be implicated in a multitude of protein complexes. Coiled coils are typically stable in long peptides (>21-28 residues), because sufficient intra- and interstrand contacts are not available in short peptides to coax strand assembly. To engineer conformationally stable CHDs in short sequences, we introduced a covalent linkage in place of an interhelical salt bridge and sculpted the helical interface with optimal hydrophobic packing. CHDs have shown efficacy for the disruption of targeted protein-protein interactions in biochemical, cellular, and animal models. This article describes our optimized approach to design and synthesize parallel and antiparallel helical tertiary structure mimics. Synthesis of CHDs involves conjugation of individual peptide segments, purification of the mono-conjugated strand, and alkylation of the two independent strands to yield crosslinked dimers. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Protocol for bis-triazole CHDs Basic Protocol 2: Protocol for dibenzyl ether CHDs.

Applicability of the nearest-neighbour model for pseudoknot RNAs.

The validity of the nearest-neighbour (NN) model was verified in an RNA pseudoknot (PK) structure. The thermodynamic parameters of the second hairpin stem (S2) region, which separates the PK from a hairpin structure, were monitored using CD and UV melting. Different PKs with identical NN base pairs in the S2 region exhibited similar thermodynamic parameters, highlighting the validity of the NN model in this RNA tertiary structure motif.

Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi‡.

Telomerase plays critical roles in cellular aging, in the emergence and/or development of cancer, and in the capacity for stem-cell renewal, consists of a catalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA (TER). TERs from diverse organisms contain two conserved structural elements: the template-pseudoknot (T-PK) and a helical three-way junction (TWJ). Species-specific features of the structure and function of telomerase make obtaining a more in-depth understanding of the molecular mechanism of telomerase particularly important. Here, we report the first structural studies of N-terminally truncated TERTs from Candida albicans and Candida tropicalis in apo form and complexed with their respective TWJs in several conformations. We found that Candida TERT proteins perform only one round of telomere addition in the presence or absence of PK/TWJ and display standard reverse transcriptase activity. The C-terminal domain adopts at least two extreme conformations and undergoes conformational interconversion, which regulates the catalytic activity. Most importantly, we identified a conserved tertiary structural motif, called the U-motif, which interacts with the reverse transcriptase domain and is crucial for catalytic activity. Together these results shed new light on the structure and mechanics of fungal TERTs, which show common TERT characteristics, but also display species-specific features.

Substitution-Inert Polynuclear Platinum Complexes Inhibit Reverse Transcription Preferentially in RNA Triplex-Forming Templates.

RNA triplexes are significant tertiary structure motifs that are found in many functional RNAs. Hence, small molecules capable of recognition, binding, and stabilization of the triple-helical RNA structures are emerging as attractive potential molecular biology tools and therapeutic agents. Here, we utilize methods of molecular biology and biophysics to study the interactions of a series of antitumor substitution-inert polynuclear platinum complexes (SI-PPCs) with triple-helical RNA structures. We show that SI-PPCs recognize and stabilize RNA triplexes and inhibit reverse transcription preferentially in the RNA template prone to the triplex formation. These so far unexplored properties of SI-PPCs suggest that the targeting of triple-stranded regions in RNA might contribute to the biological effects of SI-PPCs.

Conserved Pseudoknots in lncRNA MEG3 Are Essential for Stimulation of the p53 Pathway.

SummaryLong non-coding RNAs (lncRNAs) are key regulatory molecules, but unlike with other RNAs, the direct link between their tertiary structure motifs and their function has proven elusive. Here we report structural and functional studies of human maternally expressed gene 3 (MEG3), a tumor suppressor lncRNA that modulates the p53 response. We found that, in an evolutionary conserved region of MEG3, two distal motifs interact by base complementarity to form alternative, mutually exclusive pseudoknot structures (“kissing loops”). Mutations that disrupt these interactions impair MEG3-dependent p53 stimulation in vivo and disrupt MEG3 folding in vitro. These findings provide mechanistic insights into regulation of the p53 pathway by MEG3 and reveal how conserved motifs of tertiary structure can regulate lncRNA biological function.

Reassessment of Viroid RNA Cytosine Methylation Status at the Single Nucleotide Level.

Composed of a few hundreds of nucleotides, viroids are infectious, circular, non-protein coding RNAs able to usurp plant cellular enzymes and molecular machineries to replicate and move in their hosts. Several secondary and tertiary RNA structural motifs have been implicated in the viroid infectious cycle, but whether modified nucleotides, such as 5C-methylcytosine (m5C), also play a role has not been deeply investigated so far. Here, the possible existence of m5C in both RNA polarity strands of potato spindle tuber viroid and avocado sunblotch viroid -which are representative members of the nucleus- and chloroplast-replicating viroids, respectively- has been assessed at single nucleotide level. We show that a standard bisulfite protocol efficiently used for identifying m5C in cellular RNAs may generate false positive results in the case of the highly structured viroid RNAs. Applying a bisulfite conversion protocol specifically adapted to RNAs with high secondary structure, no m5C was identified in both polarity strands of both viroids, indicating that this specific nucleotide modification does not likely play a role in viroid biology.

Aberrant Expression of Pseudogene-Derived lncRNAs as an Alternative Mechanism of Cancer Gene Regulation in Lung Adenocarcinoma.

Transcriptome sequencing has led to the widespread identification of long non-coding RNAs (lncRNAs). Subsequently, these genes have been shown to hold functional importance in human cellular biology, which can be exploited by tumors to drive the hallmarks of cancer. Due to the complex tertiary structure and unknown binding motifs of lncRNAs, there is a growing disparity between the number of lncRNAs identified and those that have been functionally characterized. As such, lncRNAs deregulated in cancer may represent critical components of cancer pathways that could serve as novel therapeutic intervention points. Pseudogenes are non-coding DNA sequences that are defunct relatives of their protein-coding parent genes but retain high sequence similarity. Interestingly, certain lncRNAs expressed from pseudogene loci have been shown to regulate the protein-coding parent genes of these pseudogenes in trans particularly because of this sequence complementarity. We hypothesize that this phenomenon occurs more broadly than previously realized, and that aberrant expression of lncRNAs overlapping pseudogene loci provides an alternative mechanism of cancer gene deregulation. Using RNA-sequencing data from two cohorts of lung adenocarcinoma, each paired with patient-matched non-malignant lung samples, we discovered 104 deregulated pseudogene-derived lncRNAs. Remarkably, many of these deregulated lncRNAs (i) were expressed from the loci of pseudogenes related to known cancer genes, (ii) had expression that significantly correlated with protein-coding parent gene expression, and (iii) had lncRNA protein-coding parent gene expression that was significantly associated with survival. Here, we uncover evidence to suggest the lncRNA-pseudogene-protein-coding gene axis as a prominent mechanism of cancer gene regulation in lung adenocarcinoma, and highlights the clinical utility of exploring the non-coding regions of the cancer transcriptome.

Tertiary Structural Motif Sequence Statistics Enable Facile Prediction and Design of Peptides that Bind Anti-apoptotic Bfl-1 and Mcl-1

Understanding the relationship between protein sequence and structure well enough to design new proteins with desired functions is a longstanding goal in protein science. Here, we show that recurring tertiary structural motifs (TERMs) in the PDB provide rich information for protein-peptide interaction prediction and design. TERM statistics can be used to predict peptide binding energies for Bcl-2 family proteins as accurately as widely used structure-based tools. Furthermore, design using TERM energies (dTERMen) rapidly and reliably generates high-affinity peptide binders of anti-apoptotic proteins Bfl-1 and Mcl-1 with just 15%-38% sequence identity to any known native Bcl-2 family protein ligand. High-resolution structures of four designed peptides bound to their targets provide opportunities to analyze the strengths and limitations of the computational design method. Our results support dTERMen as a powerful approach that can complement existing tools for protein engineering.

Abstract A04: Altered expression of lncRNAs overlapping pseudogene loci as an alternative mechanism of cancer gene regulation

Abstract The advent of next-generation sequencing has begun to reveal the functional importance of long noncoding RNAs (lncRNAs) in human cell biology, which can be exploited by tumors to drive the hallmarks of cancer. Due to their complex tertiary structure and unknown binding motifs, there is a growing disparity between number of lncRNAs identified and those that have been functionally characterized. As such, lncRNAs deregulated in cancer may represent critical members of cancer pathways that could hold therapeutic applicability. Pseudogenes are noncoding DNA sequences that are defunct relatives of their protein-coding parent genes but retain high sequence homology. Interestingly, several lncRNAs expressed from pseudogene loci have been shown to regulate the protein-coding parent genes of these pseudogenes in trans due to sequence complementarity. We hypothesize that this phenomenon occurs more broadly than previously realized, and that aberrant expression of lncRNAs overlapping pseudogene loci provides an alternative mechanism of cancer gene deregulation. Illumina HiSeq reads were processed and aligned to the ENSEMBL annotation. Two datasets were selected due to their paired nature, complete with both lung adenocarcinoma (LUAD) and nonmalignant lung profiles (TCGA n=108, BCCA n=72). LncRNAs were filtered based on positional overlap within pseudogene loci. Differential expression was identified by Wilcoxon sign-rank test (FDR p<0.05). To identify lncRNAs that likely regulate protein-coding parent gene expression in trans, tumors were ranked by lncRNA expression, and protein-coding parent gene expression of top and bottom ranked tertiles was compared by Mann Whitney U-test (p<0.05). Survival analysis was performed using a Cox proportional hazard model. Our analysis identified 129 lncRNAs expressed from pseudogene loci that were significantly deregulated in LUAD in both datasets, many of which were correlated with clinical features such as smoking status and patient survival. Remarkably, many of these deregulated lncRNAs (i) were expressed from the loci of pseudogenes related to known cancer genes, (ii) had expression that significantly correlated with protein-coding parent gene expression, and (iii) protein-coding parent gene expression was significantly associated with survival. For example, AC008132.13 is a lncRNA expressed from a pseudogene to the B cell receptor (BCR), a previously described cancer gene involved in driving B-cell malignancies. AC008132.13 was overexpressed in LUAD and significantly positively correlated with BCR expression. In addition, BCR was significantly associated with patient survival (p=4.3e-04). This work uncovers evidence to suggest that the lncRNA-pseudogene-protein-coding gene axis is a prominent mechanism of gene deregulation exploited by lung tumors. Further characterization of this understudied gene-regulatory mechanism could reveal novel means of silencing oncogenes or reactivating tumor-suppressor genes. Citation Format: Greg L. Stewart, Katey S.S. Enfield, Victor D. Martinez, Erin A. Marshall, Stephen Lam, Wan L. Lam. Altered expression of lncRNAs overlapping pseudogene loci as an alternative mechanism of cancer gene regulation [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A04.

Monovalent ions modulate the flux through multiple folding pathways of an RNA pseudoknot

The functions of RNA pseudoknots (PKs), which are minimal tertiary structural motifs and an integral part of several ribozymes and ribonucleoprotein complexes, are determined by their structure, stability, and dynamics. Therefore, it is important to elucidate the general principles governing their thermodynamics/folding mechanisms. Here, we combine laser temperature-jump experiments and coarse-grained simulations to determine the folding/unfolding pathways of VPK, a variant of the mouse mammary tumor virus (MMTV) PK involved in ribosomal frameshifting. Fluorescent nucleotide analogs (2-aminopurine and pyrrolocytidine) placed at different stem/loop positions in the PK serve as local probes allowing us to monitor the order of assembly of VPK that has two constituent hairpins with different intrinsic stabilities. We show that at 50 mM KCl, the dominant folding pathway populates only the more stable hairpin intermediate; as the salt concentration is increased, a parallel folding pathway emerges involving the less stable hairpin as an alternate intermediate. Notably, the flux between the pathways is modulated by the ionic strength. Our findings support the principle that the order of PK structure formation is determined by the relative stabilities of the hairpins, which can be altered by sequence variations or salt concentrations. The experimental results of salt effects on the partitioning between the two folding pathways are in remarkable agreement with simulations that were performed with no adjustable parameters. Our study not only unambiguously demonstrates that VPK folds by parallel pathways but also showcases the power of combining experiments and simulations for a more enriched description of RNA self-assembly.

Hydrogen Bond Surrogate Stabilization of β-Hairpins.

Peptide secondary and tertiary structure motifs frequently serve as inspiration for the development of protein-protein interaction (PPI) inhibitors. While a wide variety of strategies have been used to stabilize or imitate α-helices, similar strategies for β-sheet stabilization are more limited. Synthetic scaffolds that stabilize reverse turns and cross-strand interactions have provided important insights into β-sheet stability and folding. However, these templates occupy regions of the β-sheet that might impact the β-sheet's ability to bind at a PPI interface. Here, we present the hydrogen bond surrogate (HBS) approach for stabilization of β-hairpin peptides. The HBS linkage replaces a cross-strand hydrogen bond with a covalent linkage, conferring significant conformational and proteolytic resistance. Importantly, this approach introduces the stabilizing linkage in the buried β-sheet interior, retains all side chains for further functionalization, and allows efficient solid-phase macrocyclization. We anticipate that HBS stabilization of PPI β-sheets will enhance the development of β-sheet PPI inhibitors and expand the repertoire of druggable PPIs.

Predicting 3D structure and stability of RNA pseudoknots in monovalent and divalent ion solutions.

RNA pseudoknots are a kind of minimal RNA tertiary structural motifs, and their three-dimensional (3D) structures and stability play essential roles in a variety of biological functions. Therefore, to predict 3D structures and stability of RNA pseudoknots is essential for understanding their functions. In the work, we employed our previously developed coarse-grained model with implicit salt to make extensive predictions and comprehensive analyses on the 3D structures and stability for RNA pseudoknots in monovalent/divalent ion solutions. The comparisons with available experimental data show that our model can successfully predict the 3D structures of RNA pseudoknots from their sequences, and can also make reliable predictions for the stability of RNA pseudoknots with different lengths and sequences over a wide range of monovalent/divalent ion concentrations. Furthermore, we made comprehensive analyses on the unfolding pathway for various RNA pseudoknots in ion solutions. Our analyses for extensive pseudokonts and the wide range of monovalent/divalent ion concentrations verify that the unfolding pathway of RNA pseudoknots is mainly dependent on the relative stability of unfolded intermediate states, and show that the unfolding pathway of RNA pseudoknots can be significantly modulated by their sequences and solution ion conditions.

Topological Constraints and Their Conformational Entropic Penalties on RNA Folds

Functional RNAs can fold into intricate structures using a number of different secondary and tertiary structural motifs. Many factors contribute to the overall free energy of the target fold. This study aims at quantifying the entropic costs coming from the loss of conformational freedom when the sugar-phosphate backbone is subjected to constraints imposed by secondary and tertiary contacts. Motivated by insights from topology theory, we design a diagrammatic scheme to represent different types of RNA structures so that constraints associated with a folded structure may be segregated into mutually independent subsets, enabling the total conformational entropy loss to be easily calculated as a sum of independent terms. We used high-throughput Monte Carlo simulations to simulate large ensembles of single-stranded RNA sequences in solution to validate the assumptions behind our diagrammatic scheme, examining the entropic costs for hairpin initiation and formation of many multiway junctions. Our diagrammatic scheme aids in the factorization of secondary/tertiary constraints into distinct topological classes and facilitates the discovery of interrelationships among multiple constraints on RNA folds. This perspective, which to our knowledge is novel, leads to useful insights into the inner workings of some functional RNA sequences, demonstrating how they might operate by transforming their structures among different topological classes.

Single-Molecule Fluorescence Reveals Commonalities and Distinctions among Natural and in Vitro-Selected RNA Tertiary Motifs in a Multistep Folding Pathway.

Decades of study of the RNA folding problem have revealed that diverse and complex structured RNAs are built from a common set of recurring structural motifs, leading to the perspective that a generalizable model of RNA folding may be developed from understanding of the folding properties of individual structural motifs. We used single-molecule fluorescence to dissect the kinetic and thermodynamic properties of a set of variants of a common tertiary structural motif, the tetraloop/tetraloop-receptor (TL/TLR). Our results revealed a multistep TL/TLR folding pathway in which preorganization of the ubiquitous AA-platform submotif precedes the formation of the docking transition state and tertiary A-minor hydrogen bond interactions form after the docking transition state. Differences in ion dependences between TL/TLR variants indicated the occurrence of sequence-dependent conformational rearrangements prior to and after the formation of the docking transition state. Nevertheless, varying the junction connecting the TL/TLR produced a common kinetic and ionic effect for all variants, suggesting that the global conformational search and compaction electrostatics are energetically independent from the formation of the tertiary motif contacts. We also found that in vitro-selected variants, despite their similar stability at high Mg2+ concentrations, are considerably less stable than natural variants under near-physiological ionic conditions, and the occurrence of the TL/TLR sequence variants in Nature correlates with their thermodynamic stability in isolation. Overall, our findings are consistent with modular but complex energetic properties of RNA structural motifs and will aid in the eventual quantitative description of RNA folding from its secondary and tertiary structural elements.

NMR experiments for the rapid identification of P=O···H-X type hydrogen bonds in nucleic acids.

Hydrogen bonds involving the backbone phosphate groups occur with high frequency in functional RNA molecules. They are often found in well-characterized tertiary structural motifs presenting powerful probes for the rapid identification of these motifs for structure elucidation purposes. We have shown recently that stable hydrogen bonds to the phosphate backbone can in principle be detected by relatively simple NMR-experiments, providing the identity of both the donor hydrogen and the acceptor phosphorous within the same experiment (Duchardt-Ferner et al., Angew Chem Int Ed Engl 50:7927-7930, 2011). However, for imino and hydroxyl hydrogen bond donor groups rapidly exchanging with the solvent as well as amino groups broadened by conformational exchange experimental sensitivity is severely hampered by extensive line broadening. Here, we present improved methods for the rapid identification of hydrogen bonds to phosphate groups in nucleic acids by NMR. The introduction of the SOFAST technique into 1H,31P-correlation experiments as well as a BEST-HNP experiment exploiting 3hJN,P rather than 2hJH,P coupling constants enables the rapid and sensitive identification of these hydrogen bonds in RNA. The experiments are applicable for larger RNAs (up to ~ 100-nt), for donor groups influenced by conformational exchange processes such as amino groups and for hydrogen bonds with rather labile hydrogens such as 2'-OH groups as well as for moderate sample concentrations. Interestingly, the size of the through-hydrogen bond scalar coupling constants depends not only on the type of the donor group but also on the structural context. The largest coupling constants were measured for hydrogen bonds involving the imino groups of protonated cytosine nucleotides as donors.

Sequence statistics of tertiary structural motifs reflect protein stability.

The Protein Data Bank (PDB) has been a key resource for learning general rules of sequence-structure relationships in proteins. Quantitative insights have been gained by defining geometric descriptors of structure (e.g., distances, dihedral angles, solvent exposure, etc.) and observing their distributions and sequence preferences. Here we argue that as the PDB continues to grow, it may become unnecessary to reduce structure into a set of elementary descriptors. Instead, it could be possible to deduce quantitative sequence-structure relationships in the context of precisely-defined complex structural motifs by mining the PDB for closely matching backbone geometries. To validate this idea, we turned to the the task of predicting changes in protein stability upon amino-acid substitution—a difficult problem of broad significance. We defined non-contiguous tertiary motifs (TERMs) around a protein site of interest and extracted sequence preferences from ensembles of closely-matching substructures in the PDB to predict mutational stability changes at the site, ΔΔGm. We demonstrate that these ensemble statistics predict ΔΔGm on par with state-of-the-art statistical and machine-learning methods on large thermodynamic datasets, and outperform these, along with a leading structure-based modeling approach, when tested in the context of unbiased diverse mutations. Further, we show that the performance of the TERM-based method is directly related to the amount of available relevant structural data, automatically improving with the growing PDB. This enables a means of estimating prediction accuracy. Our results clearly demonstrate that: 1) statistics of non-contiguous structural motifs in the PDB encode fundamental sequence-structure relationships related to protein thermodynamic stability, and 2) the PDB is now large enough that such statistics are already useful in practice, with their accuracy expected to continue increasing as the database grows. These observations suggest new ways of using structural data towards addressing problems of computational structural biology.