Geo-environmental factors' influence on mining operation: an indirect effect of managerial factors.

The purpose of the paper is to identify factors associated with operational factor (OF), geo-environmental factor (GEF), and managerial factor (MF) from the literature. After identification, the study intended to assess the impact of geo-environmental factors and managerial factors on operational factors of the mining industry. The study also tests the indirect effect of managerial factors between GEF and OF in the Indian environment. The geographical boundary of the study was 06 large mines of Odisha, Jharkhand, and Chhattisgarh of India. Three hundred and twenty nine number of purposive samples were collected via email and filtered and processed through the SPSS package. To find out the complex role and inter-relationship of GEF and MF on OF, the study adopted the structural equation modelling technique. The finding reflects that MF plays a partial mediation among GEF and OF. This phenomenon is completely novel in its field when it comes to the geo-environmental and management difficulties confronting mining operations. This research can aid managers in identifying key geological and environmental concerns in mining operations, as well as providing data for regulatory compliance. Overall, this study's findings can help management create policies and manage the environmental concerns of the mining sector. The study's findings provide important directions for future Indian mining research.

Structural insights into DNA double-strand break signaling.

Genomic integrity is most threatened by double-strand breaks, which, if left unrepaired, lead to carcinogenesis or cell death. The cell generates a network of protein-protein signaling interactions that emanate from the DNA damage which are now recognized as a rich basis for anti-cancer therapy development. Deciphering the structures of signaling proteins has been an uphill task owing to their large size and complex domain organization. Recent advances in mammalian protein expression/purification and cryo-EM-based structure determination have led to significant progress in our understanding of these large multidomain proteins. This review is an overview of the structural principles that underlie some of the key signaling proteins that function at the double-strand break site. We also discuss some plausible ideas that could be considered for future structural approaches to visualize and build a more complete understanding of protein dynamics at the break site.

Vitamin D is an endogenous partial agonist of the transient receptor potential vanilloid 1 channel.

KEY POINTS: 25-Hydroxyvitamin D (25OHD) is a partial agonist of TRPV1 whereby 25OHD can weakly activate TRPV1 yet antagonize the stimulatory effects of the full TRPV1 agonists capsaicin and oleoyl dopamine. 25OHD binds to TRPV1 within the same vanilloid binding pocket as capsaicin. 25OHD inhibits the potentiating effects of PKC-mediated TRPV1 activity. 25OHD reduces T-cell activation and trigeminal neuron calcium signalling mediated by TRPV1 activity. These results provide evidence that TRPV1 is a novel receptor for the biological actions of vitamin D in addition to the well-documented effects of vitamin D upon the nuclear vitamin D receptor. The results may have important implications for our current understanding of certain diseases where TRPV1 and vitamin D deficiency have been implicated, such as chronic pain and autoimmune diseases, such as type 1 diabetes. ABSTRACT: The capsaicin receptor TRPV1 plays an important role in nociception, inflammation and immunity and its activity is regulated by exogenous and endogenous lipophilic ligands. As vitamin D is lipophilic and involved in similar biological processes as TRPV1, we hypothesized that it directly regulates TRPV1 activity and function. Our calcium imaging and electrophysiological data demonstrate that vitamin D (25-hydroxyvitamin D (25OHD) and 1,25-hydroxyvitamin D (1,25OHD)) can weakly activate TRPV1 at physiologically relevant concentrations (100 nM). Furthermore, both 25OHD and 1,25OHD can inhibit capsaicin-induced TRPV1 activity (IC50  = 34.3 ± 0.2 and 11.5 ± 0.9 nM, respectively), but not pH-induced TRPV1 activity, suggesting that vitamin D interacts with TRPV1 in the same region as the TRPV1 agonist capsaicin. This hypothesis is supported by our in silico TRPV1 structural modelling studies, which place 25OHD in the same binding region as capsaicin. 25OHD also attenuates PKC-dependent TRPV1 potentiation via interactions with a known PKC phospho-acceptor residue in TRPV1. To provide evidence for a physiological role for the interaction of vitamin D with TRPV1, we employed two different cellular models known to express TRPV1: mouse CD4+ T-cells and trigeminal neurons. Our results indicate that 25OHD reduces TRPV1-induced cytokine release from T-cells and capsaicin-induced calcium activity in trigeminal neurons. In summary, we provide evidence that vitamin D is a novel endogenous regulator of TRPV1 channel activity that may play an important physiological role in addition to its known effects through the canonical nuclear vitamin D receptor pathway.

Genetic variation in human carboxylesterase CES1 confers resistance to hepatic steatosis.

Obesity often leads non-alcoholic fatty liver disease, insulin resistance and hyperlipidemia. Expression of carboxylesterase CES1 is positively correlated with increased lipid storage and plasma lipid concentration. Here we investigated structural and metabolic consequences of a single nucleotide polymorphism in CES1 gene that results in p.Gly143Glu amino acid substitution. We generated a humanized mouse model expressing CES1WT (control), CES1G143E and catalytically dead CES1S221A (negative control) in the liver in the absence of endogenous expression of the mouse orthologous gene. We show that the CES1G143E variant exhibits only 20% of the wild-type lipolytic activity. High-fat diet fed mice expressing CES1G143E had reduced liver and plasma triacylglycerol levels. The mechanism by which decreased CES1 activity exerts this hypolipidemic phenotype was determined to include decreased very-low density lipoprotein secretion, decreased expression of hepatic lipogenic genes and increased fatty acid oxidation as determined by increased plasma ketone bodies and hepatic mitochondrial electron transport chain protein abundance. We conclude that attenuation of human CES1 activity provides a beneficial effect on hepatic lipid metabolism. These studies also suggest that CES1 is a potential therapeutic target for non-alcoholic fatty liver disease management.

Diacylglycerol Acyltransferase 1 Is Regulated by Its N-Terminal Domain in Response to Allosteric Effectors.

Diacylglycerol acyltransferase 1 (DGAT1) is an integral membrane enzyme catalyzing the final and committed step in the acyl-coenzyme A (CoA)-dependent biosynthesis of triacylglycerol (TAG). The biochemical regulation of TAG assembly remains one of the least understood areas of primary metabolism to date. Here, we report that the hydrophilic N-terminal domain of Brassica napus DGAT1 (BnaDGAT11-113) regulates activity based on acyl-CoA/CoA levels. The N-terminal domain is not necessary for acyltransferase activity and is composed of an intrinsically disordered region and a folded segment. We show that the disordered region has an autoinhibitory function and a dimerization interface, which appears to mediate positive cooperativity, whereas the folded segment of the cytosolic region was found to have an allosteric site for acyl-CoA/CoA. Under increasing acyl-CoA levels, the binding of acyl-CoA with this noncatalytic site facilitates homotropic allosteric activation. Enzyme activation, on the other hand, is prevented under limiting acyl-CoA conditions (low acyl-CoA-to-CoA ratio), whereby CoA acts as a noncompetitive feedback inhibitor through interaction with the same folded segment. The three-dimensional NMR solution structure of the allosteric site revealed an α-helix with a loop connecting a coil fragment. The conserved amino acid residues in the loop interacting with CoA were identified, revealing details of this important regulatory element for allosteric regulation. Based on these results, a model is proposed illustrating the role of the N-terminal domain of BnaDGAT1 as a positive and negative modulator of TAG biosynthesis.

Reversible Unfolding of Rhomboid Intramembrane Proteases.

Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding.

Functional Implications of Domain Organization Within Prokaryotic Rhomboid Proteases.

Intramembrane proteases are membrane embedded enzymes that cleave transmembrane substrates. This interesting class of enzyme and its water mediated substrate cleavage mechanism occurring within the hydrophobic lipid bilayer has drawn the attention of researchers. Rhomboids are a family of ubiquitous serine intramembrane proteases. Bacterial forms of rhomboid proteases are mainly composed of six transmembrane helices that are preceded by a soluble N-terminal domain. Several crystal structures of the membrane domain of the E. coli rhomboid protease ecGlpG have been solved. Independently, the ecGlpG N-terminal cytoplasmic domain structure was solved using both NMR and protein crystallography. Despite these structures, we still do not know the structure of the full-length protein, nor do we know the functional role of these domains in the cell. This chapter will review the structural and functional roles of the different domains associated with prokaryotic rhomboid proteases. Lastly, we will address questions remaining in the field.

Exploring ligand recognition, selectivity and dynamics of TPR domains of chloroplast Toc64 and mitochondria Om64 from Arabidopsis thaliana.

The study aims to gain insight into the mode of ligand recognition by tetratricopeptide repeat (TPR) domains of chloroplast translocon at the outer envelope of chloroplast (Toc64) and mitochondrial Om64, two paralogous proteins that mediate import of proteins into chloroplast and mitochondria, respectively. Chaperone proteins associate with precursor proteins in the cytosol to maintain them in a translocation competent conformation and are recognized by Toc64 and Om64 that are located on the outer membrane of the target organelle. Heat shock proteins (Hsp70) and Hsp90 are two chaperones, which are known to play import roles in protein import. The C-termini of these chaperones are known to interact with the TPR domain of chloroplast Toc64 and mitochondrial Om64 in Arabidopsis thaliana (At). Using a molecular dynamics approach and binding energy calculations, we identify important residues involved in the interactions. Our findings suggest that the TPR domain from AtToc64 has higher affinity towards C-terminal residues of Hsp70. The interaction occurs as the terminal helices move towards each other enclosing the cradle on interaction of AtHsp70 with the TPR domain. In contrast, the TPR domain from AtOm64 does not discriminate between the C-termini of Hsp70 and Hsp90. These binding affinities are discussed with respect to our knowledge of protein targeting and specificity of protein import into endosymbiotic organelles in plant cells.

A look into DGAT1 through the EM lenses.

With the advent of modern detectors and robust structure solution pipeline, cryogenic electron microscopy has recently proved to be game changer in structural biology. Membrane proteins are challenging targets for structural biologists. This minireview focuses a membrane embedded triglyceride synthesizing machine, DGAT1. Decades of research had built the foundational knowledge on this enzyme's activity. However, recently solved cryo-EM structures of this enzyme, in apo and bound form, has provided critical mechanistic insights. The flipping of the catalytic histidine is critical of enzyme catalysis. The structures explain why the enzyme has preference to long fatty acyl chains over the short forms.

Introgression of SUB1 aggravates the susceptibility of the popular rice cultivars Swarna and Savitri to stagnant flooding.

Identification of the Sub1 gene for tolerance to flash flooding and its introgression into high-yielding rice cultivars are major targets in rice breeding for flood-prone rice agro-ecosystems for ensuring yield stability. However, knowledge is scant on the response of the modified genotypes under stagnant flooding (SF) to meet the challenge of finding a superior allele that may confer greater resilience to the plant under a stress-prone environment. In pursuance, we have tested the response of Sub1-introgression in two popular rice varieties, Swarna and Savitri to SF by comparing the biochemical factors in the control of flag leaf senescence and its primary production mechanisms of the parental lines' versus Sub1-introgressed lines. The activities of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GR), and ascorbate peroxidase (APX) increased while various parameters of primary production like total chlorophyll content, stomatal conductance (gs), normalized difference vegetation index (NDVI) and photosynthetic activity (Pn) decreased progressively with passage of time in the flag leaf of the cultivars during the post-anthesis period and SF-treatment increased the enzyme activity while depressing primary production further. Introgression of Sub1 had no influence on these activities under control conditions but widened the margin of effects under SF. It was concluded that the functional ability of flag leaf in mega rice cultivars like Swarna and Savitri decreased significantly by SF because of an ethylene-mediated promotion of senescence of the flag leaf. The enhancement of antioxidant enzyme activity by SF could not sustain the stability of primary production in the flag leaf. The introgression of the Sub1 gene made the cultivars more vulnerable to SF because the gene induced overexpression of ethylene.

Glucocorticoids Bind to SARS-CoV-2 S1 at Multiple Sites Causing Cooperative Inhibition of SARS-CoV-2 S1 Interaction With ACE2.

Dexamethasone may reduce mortality in COVID-19 patients. Whether dexamethasone or endogenous glucocorticoids, such as cortisol, biochemically interact with SARS-CoV-2 spike 1 protein (S1), or its cellular receptor ACE2, is unknown. Using molecular dynamics (MD) simulations and binding energy calculations, we identified 162 druggable pockets in various conformational states of S1 and all possible binding pockets for cortisol and dexamethasone. Through biochemical binding studies, we confirmed that cortisol and dexamethasone bind to S1. Limited proteolysis and mass spectrometry analyses validated several MD identified binding pockets for cortisol and dexamethasone on S1. Interaction assays indicated that cortisol and dexamethasone separately and cooperatively disrupt S1 interaction with ACE2, through direct binding to S1, without affecting ACE2 catalytic activity. Cortisol disrupted the binding of the mutant S1 Beta variant (E484K, K417N, N501Y) to ACE2. Delta and Omicron variants are mutated in or near identified cortisol-binding pockets in S1, which may affect cortisol binding to them. In the presence of cortisol, we find increased inhibition of S1 binding to ACE2 by an anti-SARS-CoV-2 S1 human chimeric monoclonal antibody against the receptor binding domain. Whether glucocorticoid/S1 direct interaction is an innate defence mechanism that may have contributed to mild or asymptomatic SARS-CoV-2 infection deserves further investigation.

Structural basis for recognition of transcriptional terminator structures by ProQ/FinO domain RNA chaperones.

The ProQ/FinO family of RNA binding proteins mediate sRNA-directed gene regulation throughout gram-negative bacteria. Here, we investigate the structural basis for RNA recognition by ProQ/FinO proteins, through the crystal structure of the ProQ/FinO domain of the Legionella pneumophila DNA uptake regulator, RocC, bound to the transcriptional terminator of its primary partner, the sRNA RocR. The structure reveals specific recognition of the 3' nucleotide of the terminator by a conserved pocket involving a ß-turn-α-helix motif, while the hairpin portion of the terminator is recognized by a conserved α-helical N-cap motif. Structure-guided mutagenesis reveals key RNA contact residues that are critical for RocC/RocR to repress the uptake of environmental DNA in L. pneumophila. Structural analysis and RNA binding studies reveal that other ProQ/FinO domains also recognize related transcriptional terminators with different specificities for the length of the 3' ssRNA tail.

Corrigendum to: Controlling the trade-off between spikelet number and grain filling: the hierarchy of starch synthesis in spikelets of rice panicle in relation to hormone dynamics.

The advent of dwarf statured rice varieties enabled a major breakthrough in yield and production, but raising the ceiling of genetically determined yield potential even further has been the breeding priority. Grain filling is asynchronous in the rice panicle; the inferior spikelets particularly on secondary branches of the basal part do not produce grains of a quality suitable for human consumption. Of the various strategies being considered, the control of ethylene production at anthesis has been a valuable route to potentially enhance genetic yield level of rice. The physiology underlying spikelet development has revealed spikelet position-specific ethylene levels determine the extent of grain filling, with higher levels resulting in ill-developed spikelet embodying poor endosperm starch content. To break the yield barrier, breeders have increased spikelet number per panicle in new large-panicle rice plants. However, the advantage of panicles with numerous spikelets has not resulted in enhanced yield because of poor filling of inferior spikelets. High spikelet number stimulates ethylene production and downgrading of starch synthesis, suggesting a trade-off between spikelet number and grain filling. High ethylene production in inferior spikelets suppresses expression of genes encoding endosperm starch synthesising enzymes. Hence, ethylene could be a retrograde signal that dictates the transcriptome dynamics for the cross talk between spikelet number and grain filling in the rice panicle, so attenuation of its activity may provide a solution to the problem of poor grain filling in large-panicle rice. This physiological linkage that reduces starch biosynthesis of inferior kernels is not genetically constitutive and amenable for modification through chemical, biotechnological, surgical and allelic manipulations. Studies on plant genotypes with different panicle architecture have opened up possibilities of selectively improving starch biosynthesis of inferior spikelets and thereby increasing grain yield through a physiological route.

Controlling the trade-off between spikelet number and grain filling: the hierarchy of starch synthesis in spikelets of rice panicle in relation to hormone dynamics.

The advent of dwarf statured rice varieties enabled a major breakthrough in yield and production, but raising the ceiling of genetically determined yield potential even further has been the breeding priority. Grain filling is asynchronous in the rice panicle; the inferior spikelets particularly on secondary branches of the basal part do not produce grains of a quality suitable for human consumption. Of the various strategies being considered, the control of ethylene production at anthesis has been a valuable route to potentially enhance genetic yield level of rice. The physiology underlying spikelet development has revealed spikelet position-specific ethylene levels determine the extent of grain filling, with higher levels resulting in ill-developed spikelet embodying poor endosperm starch content. To break the yield barrier, breeders have increased spikelet number per panicle in new large-panicle rice plants. However, the advantage of panicles with numerous spikelets has not resulted in enhanced yield because of poor filling of inferior spikelets. High spikelet number stimulates ethylene production and downgrading of starch synthesis, suggesting a trade-off between spikelet number and grain filling. High ethylene production in inferior spikelets suppresses expression of genes encoding endosperm starch synthesising enzymes. Hence, ethylene could be a retrograde signal that dictates the transcriptome dynamics for the cross talk between spikelet number and grain filling in the rice panicle, so attenuation of its activity may provide a solution to the problem of poor grain filling in large-panicle rice. This physiological linkage that reduces starch biosynthesis of inferior kernels is not genetically constitutive and amenable for modification through chemical, biotechnological, surgical and allelic manipulations. Studies on plant genotypes with different panicle architecture have opened up possibilities of selectively improving starch biosynthesis of inferior spikelets and thereby increasing grain yield through a physiological route.

Intrinsic disorder in the regulatory N-terminal domain of diacylglycerol acyltransferase 1 from Brassica napus.

Proteins with multifunctional regulatory domains often demonstrate structural plasticity or protein disorder, allowing the binding of multiple regulatory factors and post-translational modifications. While the importance of protein disorder is clear, it also poses a challenge for in vitro characterization. Here, we report protein intrinsic disorder in a plant molecular system, which despite its prevalence is less studied. We present a detailed biophysical characterization of the entire cytoplasmic N-terminal domain of Brassica napus diacylglycerol acyltransferase, (DGAT1), which includes an inhibitory module and allosteric binding sites. Our results demonstrate that the monomeric N-terminal domain can be stabilized for biophysical characterization and is largely intrinsically disordered in solution. This domain interacts with allosteric modulators of DGAT1, CoA and oleoyl-CoA, at micromolar concentrations. While solution scattering studies indicate conformational heterogeneity in the N-terminal domain of DGAT1, there is a small gain of secondary structure induced by ligand binding.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of universal stress protein F (YnaF) from Salmonella typhimurium.

The universal stress protein UspF (YnaF) is a small cytoplasmic bacterial protein. The expression of stress proteins is enhanced when cells are exposed to heat shock, nutrition starvation and certain other stress-inducing agents. YnaF promotes cell survival during prolonged exposure to stress and may activate a general mechanism for stress endurance. This manuscript reports preliminary crystallographic studies on YnaF from Salmonella typhimurium. The gene coding for YnaF was cloned and overexpressed and the protein was purified by Ni-NTA affinity chromatography. Purified YnaF was crystallized using vapour-diffusion and microbatch methods. The crystals belong to space group P2(1), with unit-cell parameters a = 37.51, b = 77.18, c = 56.34 A, beta = 101.8 degrees . A data set was collected to 2.5 A resolution with 94.6% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. Attempts to determine the structure are in progress.

Fluorescent Hexose Conjugates Establish Stringent Stereochemical Requirement by GLUT5 for Recognition and Transport of Monosaccharides.

The specificity characteristics of transporters can be exploited for the development of novel diagnostic therapeutic probes. The facilitated hexose transporter family (GLUTs) has a distinct set of preferences for monosaccharide substrates, and while some are expressed ubiquitously (e.g., GLUT1), others are quite tissue specific (e.g., GLUT5, which is overexpressed in some breast cancer tissues). While these differences have enabled the development of new molecular probes based upon hexose- and tissue-selective uptake, substrate design for compounds targeting these GLUT transporters has been encumbered by a limited understanding of the molecular interactions at play in hexose binding and transport. Four new fluorescently labeled hexose derivatives have been prepared, and their transport characteristics were examined in two breast cancer cell lines expressing mainly GLUTs 1, 2, and 5. Our results demonstrate, for the first time, a stringent stereochemical requirement for recognition and transport by GLUT5. 6-NBDF, in which all substituents are in the d-fructose configuration, is taken up rapidly into both cell lines via GLUT5. On the other hand, inversion of a single stereocenter at C-3 (6-NBDP), C-4 (6-NBDT), or C-5 (6-NDBS) results in selective transport via GLUT1. An in silico docking study employing the recently published GLUT5 crystal structure confirms this stereochemical dependence. This work provides insight into hexose-GLUT interactions at the molecular level and will facilitate structure-based design of novel substrates targeting individual members of the GLUT family and forms the basis of new cancer imaging or therapeutic agents.

Identification of Key Residues for Urate Specific Transport in Human Glucose Transporter 9 (hSLC2A9).

Human glucose transporter 9 (hSLC2A9) is critical in human urate homeostasis, for which very small deviations can lead to chronic or acute metabolic disorders. Human SLC2A9 is unique in that it transports hexoses as well as the organic anion, urate. This ability is in contrast to other homologous sugar transporters such as glucose transporters 1 and 5 (SLC2A1 &SLC2A5) and the xylose transporter (XylE), despite the fact that these transporters have similar protein structures. Our in silico substrate docking study has revealed that urate and fructose bind within the same binding pocket in hSLC2A9, yet with distinct orientations, and allowed us to identify novel residues for urate binding. Our functional studies confirmed that N429 is a key residue for both urate binding and transport. We have shown that cysteine residues, C181, C301 and C459 in hSLC2A9 are also essential elements for mediating urate transport. Additional data from chimæric protein analysis illustrated that transmembrane helix 7 of hSLC2A9 is necessary for urate transport but not sufficient to allow urate transport to be induced in glucose transporter 5 (hSLC2A5). These data indicate that urate transport in hSLC2A9 involves several structural elements rather than just a unique substrate binding pocket.

The Design and Structure of Outer Membrane Receptors from Peroxisomes, Mitochondria, and Chloroplasts.

The eukaryotic cell is defined by compartments that allow specialization of function. This compartmental structure generates a new concept in cell biology compared with the simpler prokaryotic cell structure, namely the specific targeting of proteins to intracellular compartments. Protein targeting is achieved by the action of specialized signals on proteins destined for organelles that are recognized by cognate receptors. An understanding of the specificity of targeting signal recognition leading to import requires an understanding of the receptor structures. Here, we focus on the structures of receptors of different import machineries located on the outer membrane of three organelles: peroxisomes, mitochondria, and chloroplasts. This review provides an overview of the structural features of outer membrane import receptors that recognize targeting signals. Finally, we briefly discuss combinatorial approaches that might aid in understanding the structural factors mediating receptor targeting signal recognition.

Ligand recognition by the TPR domain of the import factor Toc64 from Arabidopsis thaliana.

The specific targeting of protein to organelles is achieved by targeting signals being recognised by their cognate receptors. Cytosolic chaperones, bound to precursor proteins, are recognized by specific receptors of the import machinery enabling transport into the specific organelle. The aim of this study was to gain greater insight into the mode of recognition of the C-termini of Hsp70 and Hsp90 chaperones by the Tetratricopeptide Repeat (TPR) domain of the chloroplast import receptor Toc64 from Arabidopsis thaliana (At). The monomeric TPR domain binds with 1∶1 stoichiometry in similar micromolar affinity to both Hsp70 and Hsp90 as determined by isothermal titration calorimetry (ITC). Mutations of the terminal EEVD motif caused a profound decrease in affinity. Additionally, this study considered the contributions of residues upstream as alanine scanning experiments of these residues showed reduced binding affinity. Molecular dynamics simulations of the TPR domain helices upon peptide binding predicted that two helices within the TPR domain move backwards, exposing the cradle surface for interaction with the peptide. Our findings from ITC and molecular dynamics studies suggest that AtToc64_TPR does not discriminate between C-termini peptides of Hsp70 and Hsp90.