C-terminal Segment Research Articles

Template and target-site recognition by human LINE-1 in retrotransposition

The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease1. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs2. Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed reverse transcription with template RNA and target-site DNA. We report cryo-electron microscopy structures of the complete human L1 ORF2p bound to structured template RNAs and initiating cDNA synthesis. The template polyadenosine tract is recognized in a sequence-specific manner by five distinct domains. Among them, an RNA-binding domain bends the template backbone to allow engagement of an RNA hairpin stem with the L1 ORF2p C-terminal segment. Moreover, structure and biochemical reconstitutions demonstrate an unexpected target-site requirement: L1 ORF2p relies on upstream single-stranded DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break. Our research provides insights into the mechanism of ongoing transposition in the human genome and informs the engineering of retrotransposon proteins for gene therapy.

The farnesyl transferase inhibitor (FTI) lonafarnib improves nuclear morphology in ZMPSTE24-deficient fibroblasts from patients with the progeroid disorder MAD-B

ABSTRACTSeveral related progeroid disorders are caused by defective post-translational processing of prelamin A, the precursor of the nuclear scaffold protein lamin A, encoded by LMNA. Prelamin A undergoes farnesylation and additional modifications at its C-terminus. Subsequently, the farnesylated C-terminal segment is cleaved off by the zinc metalloprotease ZMPSTE24. The premature aging disorder Hutchinson Gilford progeria syndrome (HGPS) and a related progeroid disease, mandibuloacral dysplasia (MAD-B), are caused by mutations in LMNA and ZMPSTE24, respectively, that result in failure to process the lamin A precursor and accumulate permanently farnesylated forms of prelamin A. The farnesyl transferase inhibitor (FTI) lonafarnib is known to correct the aberrant nuclear morphology of HGPS patient cells and improves lifespan in children with HGPS. Importantly, and in contrast to a previous report, we show here that FTI treatment also improves the aberrant nuclear phenotypes in MAD-B patient cells with mutations in ZMPSTE24 (P248L or L425P). As expected, lonafarnib does not correct nuclear defects for cells with lamin A processing-proficient mutations. We also examine prelamin A processing in fibroblasts from two individuals with a prevalent laminopathy mutation LMNA-R644C. Despite the proximity of residue R644 to the prelamin A cleavage site, neither R644C patient cell line shows a prelamin A processing defect, and both have normal nuclear morphology. This work clarifies the prelamin A processing status and role of FTIs in a variety of laminopathy patient cells and supports the FDA-approved indication for the FTI Zokinvy for patients with processing-deficient progeroid laminopathies, but not for patients with processing-proficient laminopathies.

Insertion of GGMP repeat residues of Plasmodium falciparum Hsp70-1 in the lid of DnaK adversely impacts client recognition

Although Hsp70 is a conserved molecular chaperone, it exhibits some degree of functional specialisation across species. Features of Hsp70 regulating its functional specialisation remain to be fully established. We previously demonstrated that E. coli Hsp70 (DnaK) exhibits functional features that distinguishes it from PfHsp70-1, a canonical cytosolic Hsp70 of Plasmodium falciparum. One of the defining features of PfHsp70-1 is that it possesses GGMP repeat residues located in its C-terminal lid segment, while DnaK lacks this motif. Previously, we demonstrated that the insertion of GGMP repeat residues of PfHsp70-1 into E. coli DnaK abrogates the chaperone activity of DnaK. However, the role of the GGMP motif in regulating Hsp70 function remains to be fully understood. To explore the function of this motif, we expressed recombinant forms of wild type DnaK and its GGMP insertion motif, DnaK-G and systematically characterised the structure-function features of the two proteins using in silico analysis, biophysical approaches and an in cellulo complementation assay. Our findings demonstrated that the GGMP inserted in DnaK compromised various functional features such as nucleotide binding, allostery, substrate binding affinity and cellular proteome client selectivity. These findings thus, highlight the GGMP motif of Hsp70 as an important functional module.

An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

Autophosphorylation controls the transition between discrete functional and conformational states in protein kinases, yet the structural and molecular determinants underlying this fundamental process remain unclear. Here we show that c-terminal Tyr 530 is a de facto c-Src autophosphorylation site with slow time-resolution kinetics and a strong intermolecular component. On the contrary, activation-loop Tyr 419 undergoes faster kinetics and a cis-to-trans phosphorylation switch that controls c-terminal Tyr 530 autophosphorylation, enzyme specificity, and strikingly, c-Src non-catalytic function as a substrate. In line with this, we visualize by X-ray crystallography a snapshot of Tyr 530 intermolecular autophosphorylation. In an asymmetric arrangement of both catalytic domains, a c-terminal palindromic phospho-motif flanking Tyr 530 on the substrate molecule engages the G-loop of the active kinase adopting a position ready for entry into the catalytic cleft. Perturbation of the phospho-motif accounts for c-Src dysfunction as indicated by viral and colorectal cancer (CRC)-associated c-terminal deleted variants. We show that c-terminal residues 531 to 536 are required for c-Src Tyr 530 autophosphorylation, and such a detrimental effect is caused by the substrate molecule inhibiting allosterically the active kinase. Our work reveals a crosstalk between the activation and c-terminal segments that control the allosteric interplay between substrate- and enzyme-acting kinases during autophosphorylation.

Conformational particularities of beta-amyloid peptide 25-35

In Alzheimer's disease, beta-amyloid peptide (Ав) plays an important role in the mechanism of neurodegeneration. A small fragment of Лв(25-35) (with the sequence GSNKGAIIGLLM) is regarded to be the functional domain of Лв, responsible for its neurotoxic properties and represents the biological active region of Лв. Conformational analysis of each C-terminal segment of the peptide by the method of molecular mechanics revealed a limited number of most probable conformations and quite clearly helped to clarify what forces stabilize the structures. The obtained results showed that Лв(25-35) energetically has a propensity for adopting alpha-helix conformation of the C-terminal octapeptide segment. A molecular dynamics method was used to build a model of intramolecular mobility in the Лв(25-35) molecule. It was demonstrated that in low-energy conformations, Лв(25-35), the orientation of flexible structures of the N-terminal region with respect to the structures of the C-terminal region is different.

A Convenient Self-Removing Affinity Tag Method for the Simple Purification of Tagless Recombinant Proteins.

In this work, we describe a novel self-cleaving affinity tag technology based on a highly modified split-intein cleaving element. In this system, which has recently been commercialized by Protein Capture Science, LLC under the name iCapTagTM , the N-terminal segment of an engineered split intein is covalently immobilized onto a capture resin, while the smaller C-terminal intein segment is fused to the N-terminus of the desired target protein. The tagged target can then be expressed in an appropriate expression system, without concern for premature intein cleaving. During the purification, strong binding between the intein segments effectively captures the tagged target onto the capture resin while simultaneously generating a cleaving-competent intein complex. After unwanted impurities are washed from the resin, cleavage of the target protein is initiated by a shift of the buffer pH from 8.5 to 6.2. As a result, the highly purified tagless target protein is released from the column in the elution step. Alternately, the resin beads can be added directly to cell culture broth or lysate, allowing capture, purification and cleavage of the tagless target protein using a column-free format. These methods result in highly pure tagless target protein in a single step, and can thereby accelerate characterization and functional studies. In this work we demonstrate the single step purification of streptokinase, a fibrinolytic agent, and an engineered recombinant human hemoglobin 1.1 (rHb1.1). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression of high-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Basic Protocol 2: Purification of high-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform Alternate Protocol 1: Expression of low-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Alternate Protocol 2: Purification of low-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform.

Production of recombinant human long-acting IL-18 binding protein: inhibitory effect on ulcerative colitis in mice.

Interleukin-18 binding protein (IL-18BP) is a natural IL-18 inhibitor in vivo, which can effectively neutralize IL-18 and inhibit the inflammatory signaling pathway induced by IL-18, thus playing an anti-inflammatory role. Traditional production methods primarily rely on eukaryotic animal cell expression systems, which often entail complex processes, lower yields, and increase production costs. In this study, we present a novel approach for expressing IL-18BP fusion protein using the Escherichia coli (E. coli) system. The N-terminal segment of IL-18BP was fused with the small ubiquitin-related modifier (SUMO) tag, enabling soluble expression, while the C-terminal segment was fused with the human IgG1 Fc fragment to prolong its in vivo lifespan. Through screening, we obtained a high-expression engineering strain from a single colony and developed optimized protocols for fermentation and purification of the recombinant SUMO-IL-18BP-Fc protein. The SUMO tag was subsequently cleaved using SUMO protease, and the purified recombinant human IL-18BP-Fc (rhIL-18BP-Fc) exhibited a purity exceeding 90% with a yield of 1 g per liter of bacterial solution. The biological activities and underlying mechanisms of rhIL-18BP-Fc were evaluated using cell lines and a mouse model. Our results demonstrated that rhIL-18BP-Fc effectively inhibited IL-18-stimulated IFN-γ production in KG-1a cells in vitro and ameliorated DSS-induced ulcerative colitis in mice. In conclusion, we successfully employed the SUMO fusion system to achieve high-level production, soluble expression, and prolonged activity of rhIL-18BP-Fc in E. coli. These findings lay the groundwork for future large-scale industrial production and pharmaceutical development of rhIL-18BP-Fc protein. KEY POINTS: • Effective expression, fermentation, and purification of bioactive rhIL-18BP-Fc protein in E. coli. • The rhIL-18BP-Fc protein has a great potential for the therapy of ulcerative colitis by inhibiting the expression of inflammatory factors.

Combination of deep XLMS with deep learning reveals an ordered rearrangement and assembly of a major protein component of the vaccinia virion.

An outstanding problem in the understanding of poxvirus biology is the molecular structure of the mature virion. Via deep learning methods combined with chemical cross-linking mass spectrometry, we have addressed the structure and assembly pathway of P4a, a key poxvirus virion core component.

Characterization of cytoskeletal and structural effects of INF2 variants causing glomerulopathy and neuropathy

Focal segmental glomerulosclerosis (FSGS) is a common glomerular injury leading to end-stage renal disease. Monogenic FSGS is primarily ascribed to decreased podocyte integrity. Variants between residues 184 and 245 of INF2, an actin assembly factor, produce the monogenic FSGS phenotype. Meanwhile, variants between residues 57 and 184 cause a dual-faceted disease involving peripheral neurons and podocytes (Charcot–Marie–Tooth CMT/FSGS). To understand the molecular basis for INF2 disorders, we compared structural and cytoskeletal effects of INF2 variants classified into two subgroups: One (G73D, V108D) causes the CMT/FSGS phenotype, and the other (T161N, N202S) produces monogenic FSGS. Molecular dynamics analysis revealed that all INF2 variants show distinct flexibility compared to the wild-type INF2 and could affect stability of an intramolecular interaction between their N- and C-terminal segments. Immunocytochemistry of cells expressing INF2 variants showed fewer actin stress fibers, and disorganization of cytoplasmic microtubule arrays. Notably, CMT/FSGS variants caused more prominent changes in mitochondrial distribution and fragmentation than FSGS variants and these changes correlated with the severity of cytoskeletal disruption. Our results indicate that CMT/FSGS variants are associated with more severe global cellular defects caused by disrupted cytoskeleton-organelle interactions than are FSGS variants. Further study is needed to clarify tissue-specific pathways and/or cellular functions implicated in FSGS and CMT phenotypes

Crystal structure of the kinase domain of a receptor tyrosine kinase from a choanoflagellate, Monosiga brevicollis.

Genomic analysis of the unicellular choanoflagellate, Monosiga brevicollis (MB), revealed the remarkable presence of cell signaling and adhesion protein domains that are characteristically associated with metazoans. Strikingly, receptor tyrosine kinases, one of the most critical elements of signal transduction and communication in metazoans, are present in choanoflagellates. We determined the crystal structure at 1.95 Å resolution of the kinase domain of the M. brevicollis receptor tyrosine kinase C8 (RTKC8, a member of the choanoflagellate receptor tyrosine kinase C family) bound to the kinase inhibitor staurospaurine. The chonanoflagellate kinase domain is closely related in sequence to mammalian tyrosine kinases (~ 40% sequence identity to the human Ephrin kinase domain EphA3) and, as expected, has the canonical protein kinase fold. The kinase is structurally most similar to human Ephrin (EphA5), even though the extracellular sensor domain is completely different from that of Ephrin. The RTKC8 kinase domain is in an active conformation, with two staurosporine molecules bound to the kinase, one at the active site and another at the peptide-substrate binding site. To our knowledge this is the first example of staurospaurine binding in the Aurora A activation segment (AAS). We also show that the RTKC8 kinase domain can phosphorylate tyrosine residues in peptides from its C-terminal tail segment which is presumably the mechanism by which it transmits the extracellular stimuli to alter cellular function.

Non-canonical telomere protection role of FOXO3a of human skeletal muscle cells regulated by the TRF2-redox axis

Telomeric repeat binding factor 2 (TRF2) binds to telomeres and protects chromosome ends against the DNA damage response and senescence. Although the expression of TRF2 is downregulated upon cellular senescence and in various aging tissues, including skeletal muscle tissues, very little is known about the contribution of this decline to aging. We previously showed that TRF2 loss in myofibers does not trigger telomere deprotection but mitochondrial dysfunction leading to an increased level of reactive oxygen species. We show here that this oxidative stress triggers the binding of FOXO3a to telomeres where it protects against ATM activation, revealing a previously unrecognized telomere protective function of FOXO3a, to the best of our knowledge. We further showed in transformed fibroblasts and myotubes that the telomere properties of FOXO3a are dependent on the C-terminal segment of its CR2 domain (CR2C) but independent of its Forkhead DNA binding domain and of its CR3 transactivation domain. We propose that these non-canonical properties of FOXO3a at telomeres play a role downstream of the mitochondrial signaling induced by TRF2 downregulation to regulate skeletal muscle homeostasis and aging.

Structural basis of ELKS/Rab6B interaction and its role in vesicle capturing enhanced by liquid-liquid phase separation

ELKS proteins play a key role in organizing intracellular vesicle trafficking and targeting in both neurons and non-neuronal cells. While it is known that ELKS interacts with the vesicular traffic regulator, the Rab6 GTPase, the molecular basis governing ELKS-mediated trafficking of Rab6-coated vesicles, has remained unclear. In this study, we solved the Rab6B structure in complex with the Rab6-binding domain of ELKS1, revealing that a C-terminal segment of ELKS1 forms a helical hairpin to recognize Rab6B through a unique binding mode. We further showed that liquid-liquid phase separation (LLPS) of ELKS1 allows it to compete with other Rab6 effectors for binding to Rab6B and accumulate Rab6B-coated liposomes to the protein condensate formed by ELKS1. We also found that the ELKS1 condensate recruits Rab6B-coated vesicles to vesicle-releasing sites and promotes vesicle exocytosis. Together, our structural, biochemical, and cellular analyses suggest that ELKS1, via the LLPS-enhanced interaction with Rab6, captures Rab6-coated vesicles from the cargo transport machine for efficient vesicle release at exocytotic sites. These findings shed new light on the understanding of spatiotemporal regulation of vesicle trafficking through the interplay between membranous structures and membraneless condensates.

Structural Mechanism of Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein Receptor Complex Assembly in Lipid Bilayers Revealed by Solid-State NMR.

Synaptic vesicle fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including synaptobrevin-2 (Syb-2), syntaxin-1 (Syx-1), and SNAP-25. However, it remains controversial whether the formation of thoroughly contacted α-helical bundle from the SNARE motifs to the end of the transmembrane domains (TMDs) is necessary for SNARE-mediated membrane fusion. In this study, we characterized the conformation of Syb-2 in different assembly states using a combination of dipolar- and scalar-based solid-state NMR experiments in lipid bilayers. Our spectral analysis revealed a highly dynamic nature of the Syb-2 TMD with considerable α-helical contents. Chemical shift perturbation and mutational analysis indicated that the coupling between Syb-2 and Syx-1 TMDs mediated by residue Gly-100 of Syb-2 together with high mobility of the C-terminal segment of Syb-2 TMD are required for inner membrane merger. Our results provide new insights into the role of the Syb-2 TMD in driving membrane fusion, which improves the current understanding of the structural mechanism of SNARE complex assembly. This study highlights the significance of membrane environments in elucidating the mechanism of membrane proteins.

The periplasmic coiled coil formed by the assembly platform proteins PulL and PulM is critical for function of the Klebsiella type II secretion system

Bacteria use type II secretion systems (T2SS) to secrete to their surface folded proteins that confer diverse functions, from nutrient acquisition to virulence. In the Klebsiella species, T2SS-mediated secretion of pullulanase (PulA) requires assembly of a dynamic filament called the endopilus. The inner membrane assembly platform (AP) subcomplex is essential for endopilus assembly and PulA secretion. AP components PulL and PulM interact with each other through their C-terminal globular domains and transmembrane segments. Here, we investigated the roles of their periplasmic helices, predicted to form a coiled coil, in assembly and function of the PulL–PulM complex. PulL and PulM variants lacking these periplasmic helices were defective for interaction in the bacterial two-hybrid (BACTH) assay. Their functions in PulA secretion and assembly of PulG subunits into endopilus filaments were strongly reduced. Interestingly, deleting the cytoplasmic peptide of PulM nearly abolished the function of variant PulMΔN and its interaction with PulG, but not with PulL, in the BACTH assay. Nevertheless, PulL was specifically proteolyzed in the presence of the PulMΔN variant, suggesting that PulM N-terminal peptide stabilizes PulL in the cytoplasm. We discuss the implications of these results for the T2S endopilus and type IV pilus assembly mechanisms.

Inhibition of FAM46/TENT5 activity by BCCIPα adopting a unique fold.

The FAM46 (also known as TENT5) proteins are noncanonical poly(A) polymerases (PAPs) implicated in regulating RNA stability. The regulatory mechanisms of FAM46 are poorly understood. Here, we report that the nuclear protein BCCIPα, but not the alternatively spliced isoform BCCIPβ, binds FAM46 and inhibits their PAP activity. Unexpectedly, our structures of the FAM46A/BCCIPα and FAM46C/BCCIPα complexes show that, despite sharing most of the sequence and differing only at the C-terminal portion, BCCIPα adopts a unique structure completely different from BCCIPβ. The distinct C-terminal segment of BCCIPα supports the adoption of the unique fold but does not directly interact with FAM46. The β sheets in BCCIPα and FAM46 pack side by side to form an extended β sheet. A helix-loop-helix segment in BCCIPα inserts into the active site cleft of FAM46, thereby inhibiting the PAP activity. Our results together show that the unique fold of BCCIPα underlies its interaction with and functional regulation of FAM46.

Mutational and Environmental Effects on the Dynamic Conformational Distributions of Lys48-Linked Ubiquitin Chains.

In multidomain proteins, individual domains connected by flexible linkers are dynamically rearranged upon ligand binding and sensing changes in environmental factors, such as pH and temperature. Here, we characterize dynamic domain rearrangements of Lys48-linked ubiquitin (Ub) chains as models of multidomain proteins in which molecular surfaces mediating intermolecular interactions are involved in intramolecular domain-domain interactions. Using NMR and other biophysical techniques, we characterized dynamic conformational interconversions of diUb between open and closed states regarding solvent exposure of the hydrophobic surfaces of each Ub unit, which serve as binding sites for various Ub-interacting proteins. We found that the hydrophobic Ub-Ub interaction in diUb was reinforced by cysteine substitution of Lys48 of the distal Ub unit because of interaction between the cysteinyl thiol group and the C-terminal segment of the proximal Ub unit. In contrast, the replacement of the isopeptide linker with an artificial ethylenamine linker minimally affected the conformational distributions. Furthermore, we demonstrated that the mutational modification allosterically impacted the exposure of the most distal Ub unit in triUb. Thus, the conformational interconversion of Ub chains offers a unique design framework in Ub-based protein engineering not only for developing biosensing probes but also for allowing new opportunities for the allosteric regulation of multidomain proteins.

Structural intrinsic disorder in a functionalized potyviral coat protein as a main viability determinant of its assembled nanoparticles

The viability of viral-derived nanoparticles (virions and VLPs) aimed to nanobiotechnological functionalizations of the coat protein (CP) of turnip mosaic virus has been studied by means of advanced computational methodologies that include molecular dynamics. The study has allowed to model the structure of the complete CP and its functionalization with three different peptides and obtain essential structural features such as order/disorder, interactions, and electrostatic potentials of their constituent domains. The results provide for the first time a dynamic view of a complete potyvirus CP, since experimental available structures so far obtained lack N- and C-terminal segments. The relevance of disorder in the most distal N-terminal subdomain, and the interaction of the less distal N-terminal subdomain with the highly ordered CP core, stand out as crucial characteristic for a viable CP. Preserving them proved of outmost importance to obtain viable potyviral CPs presenting peptides at their N-terminus.

Structures of calmodulin–melittin complexes show multiple binding modes lacking classical anchoring interactions

Calmodulin (CaM) is a Ca2+ sensor protein found in all eukaryotic cells that regulates a large number of target proteins in a Ca2+ concentration-dependent manner. As a transient type hub protein, it recognizes linear motifs of its targets, though for the Ca2+-dependent binding no consensus sequence was identified. Its complex with melittin, a major component of bee venom, is often used as a model system of protein - protein complexes. Yet, the structural aspects of the binding are not well understood, as only diverse, low-resolution data are available concerning the association. We present the crystal structure of melittin in complex with Ca2+-saturated calmodulins from two, evolutionarily distant species, Homo sapiens and Plasmodium falciparum representing three binding modes of the peptide. Results - augmented by molecular dynamics simulations - indicate that multiple binding modes can exist for CaM-melittin complexes, as an intrinsic characteristic of the binding. While the helical structure of melittin remains, swapping of its salt bridges and partial unfolding of its C-terminal segment can occur. In contrast to the classical way of target recognition by CaM, we found that different sets of residues can anchor at the hydrophobic pockets of CaM, which were considered as main recognition sites. Finally, the nanomolar binding affinity of the CaM-melittin complex is created by an ensemble of arrangements of similar stability - tight binding is achieved not by optimized specific interactions but by simultaneously satisfying less optimal interaction patterns in co-existing different conformers.

Cooperation of N- and C-terminal substrate transmembrane domain segments in intramembrane proteolysis by γ-secretase

Intramembrane proteases play a pivotal role in biology and medicine, but how these proteases decode cleavability of a substrate transmembrane (TM) domain remains unclear. Here, we study the role of conformational flexibility of a TM domain, as determined by deuterium/hydrogen exchange, on substrate cleavability by γ-secretase in vitro and in cellulo. By comparing hybrid TMDs based on the natural amyloid precursor protein TM domain and an artificial poly-Leu non-substrate, we find that substrate cleavage requires conformational flexibility within the N-terminal half of the TMD helix (TM-N). Robust cleavability also requires the C-terminal TM sequence (TM-C) containing substrate cleavage sites. Since flexibility of TM-C does not correlate with cleavage efficiency, the role of the TM-C may be defined mainly by its ability to form a cleavage-competent state near the active site, together with parts of presenilin, the enzymatic component of γ-secretase. In sum, cleavability of a γ-secretase substrate appears to depend on cooperating TM domain segments, which deepens our mechanistic understanding of intramembrane proteolysis.

The C-terminal end segment of troponin T is a novel tropomyosin-binding site directly regulated by Ca2+ to determine the kinetics of cardiac muscle.

The troponin complex plays a central role in the Ca2+-regulation of striated muscle contraction and relaxation. Its inhibitory subunit TnI and tropomyosin (Tm)-binding subunit TnT are striated muscle-specific proteins evolved from a TnI-like ancestor gene. Deletion of the evolutionarily added N-terminal variable region of cardiac TnT (cTnT-ND) by restrictive proteolysis in response to myocardial energetic crisis and failure reduces ventricular peak contractile velocity to increase ejection time and increase stroke volume (J Physiol 586:3537-50, 2008). This compensatory effect corresponds to the restoration of an epitope structure recognized by a monoclonal antibody raised against the C-terminal end 27 amino acids (AA) Tm-binding segment of TnI (TnI-C27, JMCC 136:42-52, 2019). To reveal the underlying mechanism of this function of cTnT-ND, we located the conditional TnI-C27-like structure in the 14 AA C-terminal end segment of cTnT (cTnT-C14). Microplate protein binding studies and localized surface plasmon resonance analysis demonstrated that the highly conserved cTnT-C14 segment is a novel Tm-binding site besides the known Tm-binding sites 1 and 2. Quantitative data show that this cTnT-ND-enhanced third Tm-binding site of TnT functions in determining the kinetics of cardiac muscle. The molecular conformation and Tm-binding of mammalian cTnT-C14 peptide are directly regulated by physiological levels of Ca2+ with higher affinity at pCa 9 than that at pCa 4. Deletion of the last 2 AA in cTnT-C14 largely abolished Tm-binding. Cardiomyopathy mutation R286C significantly increases cTnT-C14's binding affinity for Tm at pCa 9 whereas mutation R278C increases Tm-binding with diminished Ca2+ regulation. These findings provide novel insights into the regulation of striated muscle contraction and relaxation with physiological and pathophysiological significances and identify a myofilament mechanism to improve cardiac function and treat heart failure.