Caenorhabditis elegans telomere-binding proteins TEBP-1 and TEBP-2 adapt the Myb module to dimerize and bind telomeric DNA.

Protecting chromosome ends from misrecognition as double-stranded (ds) DNA breaks is fundamental to eukaryotic viability. The protein complex shelterin prevents a DNA damage response at mammalian telomeres. Mammalian shelterin proteins TRF1 and TRF2 and their homologs in yeast and protozoa protect telomeric dsDNA. N-terminal homodimerization and C-terminal Myb-domain-mediated dsDNA binding are two structural hallmarks of end protection by TRF homologs. Yet our understanding of how Caenorhabditis elegans protects its telomeric dsDNA is limited. Recently identified C. elegans proteins TEBP-1 (also called DTN-1) and TEBP-2 (also called DTN-2) are functional homologs of TRF proteins, but how they bind DNA and whether or how they dimerize is not known. TEBP-1 and TEBP-2 harbor three Myb-containing domains (MCDs) and no obvious dimerization domain. We demonstrate biochemically that only the third MCD binds DNA. We solve the X-ray crystal structure of TEBP-2 MCD3 with telomeric dsDNA to reveal the structural mechanism of telomeric dsDNA protection in C. elegans. Mutagenesis of the DNA-binding site of TEBP-1 and TEBP-2 compromises DNA binding in vitro, and increases DNA damage signaling, lengthens telomeres, and decreases brood size in vivo. Via an X-ray crystal structure, biochemical validation of the dimerization interface, and SEC-MALS analysis, we demonstrate that MCD1 and MCD2 form a composite dimerization module that facilitates not only TEBP-1 and TEBP-2 homodimerization but also heterodimerization. These findings provide fundamental insights into C. elegans telomeric dsDNA protection and highlight how different eukaryotes have evolved distinct strategies to solve the chromosome end protection problem.

Modified Peptide Molecules As Potential Modulators of Shelterin Protein Functions; TRF1.

In this work, we present studies on relatively new and still not well-explored potential anticancer targets which are shelterin proteins, in particular the TRF1 protein can be blocked by in silico designed "peptidomimetic" molecules. TRF1 interacts directly with the TIN2 protein, and this protein-protein interaction is crucial for the proper functioning of telomere, which could be blocked by our novel modified peptide molecules. Our chemotherapeutic approach is based on assumption that modulation of TRF1-TIN2 interaction may be more harmful for cancer cells as cancer telomeres are more fragile than in normal cells. We have shown in vitro within SPR experiments that our modified peptide PEP1 molecule interacts with TRF1, presumably at the site originally occupied by the TIN2 protein. Disturbance of the shelterin complex by studied molecule may not in short term lead to cytotoxic effects, however blocking TRF1-TIN2 resulted in cellular senescence in cellular breast cancer lines used as a cancer model. Thus, our compounds appeared useful as starting model compounds for precise blockage of TRF proteins.

EPAS1 prevents telomeric damage-induced senescence by enhancing transcription of TRF1, TRF2, and RAD50.

Telomeres are nucleoprotein structures located at the end of each chromosome, which function in terminal protection and genomic stability. Telomeric damage is closely related to replicative senescence in vitro and physical aging in vivo. As relatively long-lived mammals based on body size, bats display unique telomeric patterns, including the up-regulation of genes involved in alternative lengthening of telomeres (ALT), DNA repair, and DNA replication. At present, however, the relevant molecular mechanisms remain unclear. In this study, we performed cross-species comparison and identified EPAS1, a well-defined oxygen response gene, as a key telomeric protector in bat fibroblasts. Bat fibroblasts showed high expression of EPAS1, which enhanced the transcription of shelterin components TRF1 and TRF2, as well as DNA repair factor RAD50, conferring bat fibroblasts with resistance to senescence during long-term consecutive expansion. Based on a human single-cell transcriptome atlas, we found that EPAS1 was predominantly expressed in the human pulmonary endothelial cell subpopulation. Using in vitro-cultured human pulmonary endothelial cells, we confirmed the functional and mechanistic conservation of EPAS1 in telomeric protection between bats and humans. In addition, the EPAS1 agonist M1001 was shown to be a protective compound against bleomycin-induced pulmonary telomeric damage and senescence. In conclusion, we identified a potential mechanism for regulating telomere stability in human pulmonary diseases associated with aging, drawing insights from the longevity of bats.

Assembly path dependence of telomeric DNA compaction by TRF1, TIN2, and SA1.

Telomeres, complexes of DNA and proteins, protect ends of linear chromosomes. In humans, the two shelterin proteins TRF1 and TIN2, along with cohesin subunit SA1, were proposed to mediate telomere cohesion. Although the ability of the TRF1-TIN2 and TRF1-SA1 systems to compact telomeric DNA by DNA-DNA bridging has been reported, the function of the full ternary TRF1-TIN2-SA1 system has not been explored in detail. Here, we quantify the compaction of nanochannel-stretched DNA by the ternary system, as well as its constituents, and obtain estimates of the relative impact of its constituents and their interactions. We find that TRF1, TIN2, and SA1 work synergistically to cause a compaction of the DNA substrate, and that maximal compaction occurs if all three proteins are present. By altering the sequence with which DNA substrates are exposed to proteins, we establish that compaction by TRF1 and TIN2 can proceed through binding of TRF1 to DNA, followed by compaction as TIN2 recognizes the previously bound TRF1. We further establish that SA1 alone can also lead to a compaction, and that compaction in a combined system of all three proteins can be understood as an additive effect of TRF1-TIN2 and SA1-based compaction. Atomic force microscopy of intermolecular aggregation confirms that a combination of TRF1, TIN2, and SA1 together drive strong intermolecular aggregation as it would be required during chromosome cohesion.

In Vitro Characterization of the Physical Interactions between the Long Noncoding RNA TERRA and the Telomeric Proteins TRF1 and TRF2.

RNA-protein interactions drive key cellular pathways such as protein translation, nuclear organization and genome stability maintenance. The human telomeric protein TRF2 binds to the long noncoding RNA TERRA through independent domains, including its N-terminal B domain. We previously demonstrated that TRF2 B domain binding to TERRA supports invasion of TERRA into telomeric double stranded DNA, leading to the formation of telomeric RNA:DNA hybrids. The other telomeric protein TRF1, which also binds to TERRA, suppresses this TRF2-associated activity by preventing TERRA-B domain interactions. Herein, we show that the binding of both TRF1 and TRF2 to TERRA depends on the ability of the latter to form G-quadruplex structures. Moreover, a cluster of arginines within the B domain is largely responsible for its binding to TERRA. On the other side, a patch of glutamates within the N-terminal A domain of TRF1 mainly accounts for the inhibition of TERRA-B domain complex formation. Finally, mouse TRF2 B domain binds to TERRA, similarly to its human counterpart, while mouse TRF1 A domain lacks the inhibitory activity. Our data shed further light on the complex crosstalk between telomeric proteins and RNAs and suggest a lack of functional conservation in mouse.

Impact of non-synonymous mutations on the structure and function of telomeric repeat binding factor 1.

Telomeric repeat binding factor 1 (TRF1) is one of the major components of the shelterin complex. It directly binds to the telomere and controls its function by regulating the telomerase acting on it. Several variations are reported in the TRF1 gene; some are associated with variety of diseases. Here, we have studied the structural and functional significance of these variations in the TRFH domain of TRF1. We have used cutting-edge computational methods such as SIFT, PolyPhen-2, PROVEAN, Mutation Assessor, mCSM, SDM, STRUM, MAESTRO, and DUET to predict the effects of 124 mutations in the TRFH domain of TRF1. Out of 124 mutations, we have identified 12 deleterious mutations with high confidence based on their prediction. To see the impact of the finally selected mutations on the structure and stability of TRF1, all-atom molecular dynamics (MD) simulations on TRF1-Wild type (WT), L79R and P150R mutants for 200 ns were carried out. A significant conformational change in the structure of the P150R mutant was observed. Our integrated computational study provides a comprehensive understanding of structural changes in TRF1 incurred due to the mutations and subsequent function, leading to the progression of many diseases.Communicated by Ramaswamy H. Sarma.

Transcriptional regulation of telomeric repeat-containing RNA by acridine derivatives.

Telomere is a specialized DNA-protein complex that plays an important role in maintaining chromosomal integrity. Shelterin is a protein complex formed by six different proteins, with telomeric repeat factors 1 (TRF1) and 2 (TRF2) binding to double-strand telomeric DNA. Telomeric DNA consists of complementary G-rich and C-rich repeats, which could form G-quadruplex and intercalated motif (i-motif), respectively, during cell cycle. Its G-rich transcription product, telomeric repeat-containing RNA (TERRA), is essential for telomere stability and heterochromatin formation. After extensive screening, we found that acridine derivative 2c and acridine dimer DI26 could selectively interact with TRF1 and telomeric i-motif, respectively. Compound 2c blocked the binding of TRF1 with telomeric duplex DNA, resulting in up-regulation of TERRA. Accumulated TERRA could bind with TRF1 at its allosteric site and further destabilize its binding with telomeric DNA. In contrast, DI26 could destabilize telomeric i-motif, resulting in down-regulation of TERRA. Both compounds exhibited anti-tumour activity for A549 cells, but induced different DNA damage pathways. Compound 2c significantly suppressed tumour growth in A549 xenograft mouse model. The function of telomeric i-motif structure was first studied with a selective binding ligand, which could play an important role in regulating TERRA transcription. Our results showed that appropriate level of TERRA transcript could be important for stability of telomere, and acridine derivatives could be further developed as anti-cancer agents targeting telomere. This research increased understanding for biological roles of telomeric i-motif, TRF1 and TERRA, as potential anti-cancer drug targets.

Degradation of a Novel DNA Damage Response Protein, Tankyrase 1 Binding Protein 1, following Adenovirus Infection.

Infection by most DNA viruses activates a cellular DNA damage response (DDR), which may be to the detriment or advantage of the virus. In the case of adenoviruses, they neutralize antiviral effects of DDR activation by targeting a number of proteins for rapid proteasome-mediated degradation. We have now identified a novel DDR protein, tankyrase 1 binding protein 1 (TNKS1BP1) (also known as Tab182), which is degraded during infection by adenovirus serotype 5 and adenovirus serotype 12. In both cases, degradation requires the action of the early region 1B55K (E1B55K) and early region 4 open reading frame 6 (E4orf6) viral proteins and is mediated through the proteasome by the action of cullin-based cellular E3 ligases. The degradation of Tab182 appears to be serotype specific, as the protein remains relatively stable following infection with adenovirus serotypes 4, 7, 9, and 11. We have gone on to confirm that Tab182 is an integral component of the CNOT complex, which has transcriptional regulatory, deadenylation, and E3 ligase activities. The levels of at least 2 other members of the complex (CNOT3 and CNOT7) are also reduced during adenovirus infection, whereas the levels of CNOT4 and CNOT1 remain stable. The depletion of Tab182 with small interfering RNA (siRNA) enhances the expression of early region 1A proteins (E1As) to a limited extent during adenovirus infection, but the depletion of CNOT1 is particularly advantageous to the virus and results in a marked increase in the expression of adenovirus early proteins. In addition, the depletion of Tab182 and CNOT1 results in a limited increase in the viral DNA level during infection. We conclude that the cellular CNOT complex is a previously unidentified major target for adenoviruses during infection.IMPORTANCE Adenoviruses target a number of cellular proteins involved in the DNA damage response for rapid degradation. We have now shown that Tab182, which we have confirmed to be an integral component of the mammalian CNOT complex, is degraded following infection by adenovirus serotypes 5 and 12. This requires the viral E1B55K and E4orf6 proteins and is mediated by cullin-based E3 ligases and the proteasome. In addition to Tab182, the levels of other CNOT proteins are also reduced during adenovirus infection. Thus, CNOT3 and CNOT7, for example, are degraded, whereas CNOT4 and CNOT1 are not. The siRNA-mediated depletion of components of the complex enhances the expression of adenovirus early proteins and increases the concentration of viral DNA produced during infection. This study highlights a novel protein complex, CNOT, which is targeted for adenovirus-mediated protein degradation. To our knowledge, this is the first time that the CNOT complex has been identified as an adenoviral target.

The structurally similar TRFH domain of TRF1 and TRF2 dimers shows distinct behaviour towards TIN2.

The telomere repeat binding-factor 1 and 2 (TRF1 and TRF2) proteins of the shelterin complex bind to duplex telomeric DNA as homodimers, and the homodimerization is mediated by their TRFH (TRF-homology) domains. We performed molecular dynamic (MD) simulations of the dimer forms of TRF1TRFH and TRF2TRFH in the presence/absence of the TIN2TBM (TIN2, TRF-interacting nuclear protein 2, TBM, TRF-binding motif) peptide. The MD results suggest that TIN2TBM is necessary to ensure the stability of TRF1TRFH homodimer but not the TRF2TRFH homodimer. In TRF1-TIN2-TRF2 complex, the peptide enhances the protein-protein interactions to yield a stable heterodimer. Both monomers in TRF1TRFH homodimer interact almost equally with the peptide, whereas in TRF2TRFH homodimer, monomer TRF2TRFH(M1) exhibits more dominant interactions than the TRF2TRFH(M2). The common residues of TRF1/2TRFH(M1) that form interactions with TIN2TBM in all peptide-bound systems originate from the H3 (helix) and L3 (loop) regions. Additionally, in the homodimer systems, residues of TRF1/2TRFH(M2) also interact with the peptide. The residue pair E71-K213 is responsible for different conformations of TRF1TRFH homodimers; specifically, this residue pair enhances the protein-peptide/protein interactions in peptide-bound/unbound systems, respectively. TRF1TRFH and TRF2TRFH proteins have a conserved but different interface responsible for the protein-protein/peptide interactions that exist in the corresponding dimers.

TRF1 participates in chromosome end protection by averting TRF2-dependent telomeric R loops.

The shelterin protein TRF2 assembles protective T loops at chromosome ends by stimulating intramolecular invasion of the telomeric G-rich single-stranded DNA (ssDNA) overhang into the duplex telomeric array. The other shelterin factor, TRF1, is thought to mainly facilitate telomeric dsDNA replication without directly participating in end protection. Here we show that in vitro human TRF2 stimulates invasion of G-rich TERRA-like RNA into telomeric dsDNA, leading to formation of telomeric RNA-DNA hybrids (telR loops). The N-terminal basic domain of TRF2 binds to TERRA-like RNA and enables TRF2 to promote efficient RNA invasion. TRF1, through its N-terminal acidic domain, counteracts TRF2-mediated RNA invasion but not ssDNA invasion. In vivo, when TRF1 is depleted or replaced with a variant lacking the acidic domain, TRF2 induces formation of telR loops, which in turn cause telomere loss. Hence, uncontrolled TRF2 threatens telomere integrity, and TRF1 directly supports end protection by suppressing harmful telR loops.

Aurora Kinase B, a novel regulator of TERF1 binding and telomeric integrity.

AURKB (Aurora Kinase B) is a serine/threonine kinase better known for its role at the mitotic kinetochore during chromosome segregation. Here, we demonstrate that AURKB localizes to the telomeres in mouse embryonic stem cells, where it interacts with the essential telomere protein TERF1. Loss of AURKB function affects TERF1 telomere binding and results in aberrant telomere structure. In vitro kinase experiments successfully identified Serine 404 on TERF1 as a putative AURKB target site. Importantly, in vivo overexpression of S404-TERF1 mutants results in fragile telomere formation. These findings demonstrate that AURKB is an important regulator of telomere structural integrity.

Conformational heterogeneity in the Hsp70 chaperone-substrate ensemble identified from analysis of NMR-detected titration data.

The Hsp70 chaperone system plays a critical role in cellular homeostasis by binding to client protein molecules. We have recently shown by methyl-TROSY NMR methods that the Escherichia coli Hsp70, DnaK, can form multiple bound complexes with a small client protein, hTRF1. In an effort to characterize the interactions further we report here the results of an NMR-based titration study of hTRF1 and DnaK, where both molecular components are monitored simultaneously, leading to a binding model. A central finding is the formation of a previously undetected 3:1 hTRF1-DnaK complex, suggesting that under heat shock conditions, DnaK might be able to protect cytosolic proteins whose net concentrations would exceed that of the chaperone. Moreover, these results provide new insight into the heterogeneous ensemble of complexes formed by DnaK chaperones and further emphasize the unique role of NMR spectroscopy in obtaining information about individual events in a complex binding scheme by exploiting a large number of probes that report uniquely on distinct binding processes.

Promiscuous binding by Hsp70 results in conformational heterogeneity and fuzzy chaperone-substrate ensembles.

The Hsp70 chaperone system is integrated into a myriad of biochemical processes that are critical for cellular proteostasis. Although detailed pictures of Hsp70 bound with peptides have emerged, correspondingly detailed structural information on complexes with folding-competent substrates remains lacking. Here we report a methyl-TROSY based solution NMR study showing that the Escherichia coli version of Hsp70, DnaK, binds to as many as four distinct sites on a small 53-residue client protein, hTRF1. A fraction of hTRF1 chains are also bound to two DnaK molecules simultaneously, resulting in a mixture of DnaK-substrate sub-ensembles that are structurally heterogeneous. The interactions of Hsp70 with a client protein at different sites results in a fuzzy chaperone-substrate ensemble and suggests a mechanism for Hsp70 function whereby the structural heterogeneity of released substrate molecules enables them to circumvent kinetic traps in their conformational free energy landscape and fold efficiently to the native state.

How proteins bind to DNA: target discrimination and dynamic sequence search by the telomeric protein TRF1.

Target search as performed by DNA-binding proteins is a complex process, in which multiple factors contribute to both thermodynamic discrimination of the target sequence from overwhelmingly abundant off-target sites and kinetic acceleration of dynamic sequence interrogation. TRF1, the protein that binds to telomeric tandem repeats, faces an intriguing variant of the search problem where target sites are clustered within short fragments of chromosomal DNA. In this study, we use extensive (>0.5 ms in total) MD simulations to study the dynamical aspects of sequence-specific binding of TRF1 at both telomeric and non-cognate DNA. For the first time, we describe the spontaneous formation of a sequence-specific native protein-DNA complex in atomistic detail, and study the mechanism by which proteins avoid off-target binding while retaining high affinity for target sites. Our calculated free energy landscapes reproduce the thermodynamics of sequence-specific binding, while statistical approaches allow for a comprehensive description of intermediate stages of complex formation.

Longitudinal relaxation optimized amide 1H-CEST experiments for studying slow chemical exchange processes in fully protonated proteins.

Chemical Exchange Saturation Transfer (CEST) experiments are increasingly used to study slow timescale exchange processes in biomolecules. Although 15N- and 13C-CEST have been the approaches of choice, the development of spin state selective 1H-CEST pulse sequences that separate the effects of chemical and dipolar exchange [T. Yuwen, A. Sekhar and L. E. Kay, Angew Chem Int Ed Engl 2016 doi: 10.1002/anie.201610759 (Yuwen et al. 2017)] significantly increases the utility of 1H-based experiments. Pulse schemes have been described previously for studies of highly deuterated proteins. We present here longitudinal-relaxation optimized amide 1H-CEST experiments for probing chemical exchange in protonated proteins. Applications involving a pair of proteins are presented establishing that accurate 1H chemical shifts of sparsely populated conformers can be obtained from simple analyses of 1H-CEST profiles. A discussion of the inherent differences between 15N-/13C- and 1H-CEST experiments is presented, leading to an optimal strategy for recording 1H-CEST experiments.

WRN is recruited to damaged telomeres via its RQC domain and tankyrase1-mediated poly-ADP-ribosylation of TRF1.

Werner syndrome (WS) is a progeroid-like syndrome caused by WRN gene mutations. WS cells exhibit shorter telomere length compared to normal cells, but it is not fully understood how WRN deficiency leads directly to telomere dysfunction. By generating localized telomere-specific DNA damage in a real-time fashion and a dose-dependent manner, we found that the damage response of WRN at telomeres relies on its RQC domain, which is different from the canonical damage response at genomic sites via its HRDC domain. We showed that in addition to steady state telomere erosion, WRN depleted cells are also sensitive to telomeric damage. WRN responds to site-specific telomeric damage via its RQC domain, interacting at Lysine 1016 and Phenylalanine1037 with the N-terminal acidic domain of the telomere shelterin protein TRF1 and demonstrating a novel mechanism for WRN's role in telomere protection. We also found that tankyrase1-mediated poly-ADP-ribosylation of TRF1 is important for both the interaction between WRN and TRF1 and the damage recruitment of WRN to telomeres. Mutations of potential tankyrase1 ADP-ribosylation sites within the RGCADG motif of TRF1 strongly diminish the interaction with WRN and the damage response of WRN only at telomeres. Taken together, our results reveal a novel mechanism as to how WRN protects telomere integrity from damage and telomere erosion.

Molecular basis and quantitative assessment of TRF1 and TRF2 protein interactions with TIN2 and Apollo peptides.

Shelterin is a six-protein complex (TRF1, TRF2, POT1, RAP1, TIN2, and TPP1) that also functions in smaller subsets in regulation and protection of human telomeres. Two closely related proteins, TRF1 and TRF2, make high-affinity contact directly with double-stranded telomeric DNA and serve as a molecular platform. Protein TIN2 binds to TRF1 and TRF2 dimer-forming domains, whereas Apollo makes interaction only with TRF2. To elucidate the molecular basis of these interactions, we employed molecular dynamics (MD) simulations of TRF1TRFH-TIN2TBM and TRF2TRFH-TIN2TBM/ApolloTBM complexes and of the isolated proteins. MD enabled a structural and dynamical comparison of protein-peptide complexes including H-bond interactions and interfacial residues that may regulate TRF protein binding to the given peptides, especially focusing on interactions described in crystallographic data. Residues with a selective function in both TRF1TRFH and TRF2TRFH and forming a stable hydrogen bond network with TIN2TBM or ApolloTBM peptides were traced. Our study revealed that TIN2TBM forms a well-defined binding mode with TRF1TRFH as compared to TRF2TRFH, and that the binding pocket of TIN2TBM is deeper for TRF2TRFH protein than ApolloTBM. The MD data provide a basis for the reinterpretation of mutational data obtained in crystallographic work for the TRF proteins. Together, the previously determined X-ray structure and our MD provide a detailed view of the TRF-peptide binding mode and the structure of TRF1/2 binding pockets. Particular TRF-peptide interactions are very specific for the formation of each protein-peptide complex, identifying TRF proteins as potential targets for the design of inhibitors/drugs modulating telomere machinery for anticancer therapy.

TRF1 phosphorylation on T271 modulates telomerase-dependent telomere length maintenance as well as the formation of ALT-associated PML bodies.

TRF1, a component of the shelterin complex, plays a key role in both telomerase-dependent telomere maintenance and alternative lengthening of telomeres, the latter also known as ALT. Characteristics of ALT cells include C-circles and ALT-associated PML bodies, referred to as APBs. The function of TRF1 is tightly regulated by post-translational modification including phosphorylation, however TRF1 phosphorylation sites have yet to be fully characterized. Here we report a novel TRF1 phosphorylation site threonine 271. We show that a nonphosphorylatable mutation of T271A impairs TRF1 binding to telomeric DNA in vivo and renders TRF1 defective in inhibiting telomerase-dependent telomere elongation. On the other hand, TRF1 carrying a phosphomimic mutation of T271D is competent in not only binding to telomeric DNA but also inhibiting telomerase-mediated telomere lengthening. These results suggest that TRF1 phosphorylation on T271 negatively regulates telomerase-mediated telomere maintenance. We find that in telomerase-negative ALT cells, TRF1 carrying either a T271A or T271D mutation is able to promote C-circle production but fails to support APB formation. These results suggest that TRF1 phosphorylation on T271 is necessary for APB formation but dispensable for C-circle production. These results further imply that APB formation can be mechanistically separated from C-circle production.

Molecular Architecture of Full-length TRF1 Favors Its Interaction with DNA.

Telomeres are specific DNA-protein structures found at both ends of eukaryotic chromosomes that protect the genome from degradation and from being recognized as double-stranded breaks. In vertebrates, telomeres are composed of tandem repeats of the TTAGGG sequence that are bound by a six-subunit complex called shelterin. Molecular mechanisms of telomere functions remain unknown in large part due to lack of structural data on shelterins, shelterin complex, and its interaction with the telomeric DNA repeats. TRF1 is one of the best studied shelterin components; however, the molecular architecture of the full-length protein remains unknown. We have used single-particle electron microscopy to elucidate the structure of TRF1 and its interaction with telomeric DNA sequence. Our results demonstrate that full-length TRF1 presents a molecular architecture that assists its interaction with telometic DNA and at the same time makes TRFH domains accessible to other TRF1 binding partners. Furthermore, our studies suggest hypothetical models on how other proteins as TIN2 and tankyrase contribute to regulate TRF1 function.

Hsp70 biases the folding pathways of client proteins.

The 70-kDa heat shock protein (Hsp70) family of chaperones bind cognate substrates to perform a variety of different processes that are integral to cellular homeostasis. Although detailed structural information is available on the chaperone, the structural features of folding competent substrates in the bound form have not been well characterized. Here we use paramagnetic relaxation enhancement (PRE) NMR spectroscopy to probe the existence of long-range interactions in one such folding competent substrate, human telomere repeat binding factor (hTRF1), which is bound to DnaK in a globally unfolded conformation. We show that DnaK binding modifies the energy landscape of the substrate by removing long-range interactions that are otherwise present in the unbound, unfolded conformation of hTRF1. Because the unfolded state of hTRF1 is only marginally populated and transiently formed, it is inaccessible to standard NMR approaches. We therefore developed a (1)H-based CEST experiment that allows measurement of PREs in sparse states, reporting on transiently sampled conformations. Our results suggest that DnaK binding can significantly bias the folding pathway of client substrates such that secondary structure forms first, followed by the development of longer-range contacts between more distal parts of the protein.