Conformational Dynamics of the Mitochondrial TIM23 Preprotein Translocase

The vast majority of mitochondrial proteins are synthesized by the cytosolic ribosomes as precursor proteins which have to be transported into the organelle to reach their sites of function. The whole process of recognition, translocation, intra-mitochondrial sorting of and assembly of precursor proteins is achieved by the concerted action of different mitochondrial translocases. All proteins destined for the mitochondrial matrix and some inner membrane proteins are imported first by the TOM complex of the outer membrane and subsequently by the TIM23 complex of the inner membrane in an energy-driven process. The TIM23 complex was found to consist of ten components, conventionally divided into two sectors: membrane sector harbouring the translocation channel and the import motor on the matrix side of the membrane sector. In the first part of the present work, the two most recently discovered subunits of the TIM23 complex, Pam17 and Tim21 were characterized. A systematic characterization revealed that both of these non-essential subunits of the translocase are associated with Tim17-Tim23 core of the membrane sector of the TIM23 translocase. A functional connection between the two non-essential components was discovered. Results presented in this part showed that Pam17 and Tim21 modulate the functions of the TIM23 complex in an antagonistic manner. The second part of the work was directed towards understanding the motor sector of the translocase in terms of the regulated interaction between Tim44 and Ssc1. Previous studies on the Tim44:Ssc1 interaction were able to discern the steady-state properties of Tim44:Ssc1 interaction in organello and in vitro. However, due to the limitations of the techniques used, they were unable to shed light on the kinetics and dynamics of the process. The translocation event is a dynamic event with conformational cycling of the various components. Therefore, the kinetic components essential in defining the cycle of events in the motor sector were explored. A FRET based assay to analyze the Tim44:Ssc1 interaction in real time was developed. The same set of tools was also used to resolve the regions of the two proteins that determine their interaction. The substrate induced dissociation of Tim44:Ssc1 complex was found to be too slow to support a physiological rate of protein translocation. ATP-induced dissociation was observed to be fast enough to be physiologically relevant. The dissociation of Ssc1 from Tim44 occurred in a one step manner without Tim44 anchored conformational changes. Furthermore, peptide-array scanning of mitochondrial matrix proteins revealed that Ssc1 and Tim44 share complementary binding sites on the precursor proteins which could prevent backsliding of preproteins. The data support the Brownian ratchet model mediated translocation of preproteins into the mitochondrial matrix. The third part of the work aimed at dissecting the chaperone cycle of Ssc1 in the mitochondrial matrix, in terms of conformational changes and binding of co-chaperones. Using the FRET sensors developed, the inter-domain conformation and lid-base conformations of the PBD of Ssc1 could be investigated. Single particle FRET (SpFRET) analysis showed that in the ATP-bound form Ssc1 populates a homogeneous conformational state with respect to the inter-domain conformation and conformation of the lid to base of the PBD. On the contrary, in the ADP-bound state the conformation of the chaperone is heterogenous. Using the same sensors on bacterial homologue DnaK, specific differences in conformational distributions were observed. Furthermore, the active role of substrates in determining the inter-domain conformation and lid-closing was evident from the SpFRET based conformational analyses. Using ensemble time resolved FRET, the kinetics and dynamics of conformational changes along with binding of co-chaperones were explored. This provided a better understanding of the conformational dynamics of Ssc1 in the context of functional chaperone cycle in the mitochondrial matrix

Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein

The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins

Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein

Abstract The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins

Understanding the effect of locked nucleic acid and 2′-O-methyl modification on the hybridization thermodynamics of a miRNA–mRNA pair in the presence and absence of AfPiwi protein

miRNAs are some of the key epigenetic regulators of gene expression. They act through hybridization with their target mRNA and modulate the level of respective proteins via different mechanisms. Various cancer conditions are known to be associated with up- and downregulation of the oncogenic and tumor suppressor miRNAs, respectively. The levels of aberrantly expressed oncogenic miRNAs can be downregulated in different ways. Similarly, restoration of tumor suppressor miRNAs to their normal levels can be achieved using miRNA mimics. However, the use of miRNA mimics is limited by their reduced biostability and function. We have studied the hybridization thermodynamics of the miRNA 26a (11-mer, including the seed sequence) guide strand with the mRNA (11-mer) target strand in the absence and presence of AfPiwi protein. We have also inserted locked nucleic acids (LNAs) and 2′-O-methyl-modified nucleotides into the guide strand, in a walk-through manner, to assess their effect on the binding efficiency between guide and target RNA. Insertion of LNA and 2′-O-methyl-modified nucleotides into the guide strand helped to strengthen the binding affinity irrespective of the position of insertion. However, in the presence of AfPiwi protein, these modifications reduced the binding affinity to different extents depending on the position of insertion. Insertion of a modification leads to an increase in the enthalpic contribution with an increased unfavorable entropic contribution, which negatively compensates for the higher favorable enthalpy

Unique structural modulation of a non-native substrate by cochaperone DnaJ

The role of bacterial DnaJ protein as a cochaperone of DnaK is strongly appreciated. Although DnaJ unaccompanied by DnaK can bind unfolded as well as native substrate proteins, its role as an individual chaperone remains elusive. In this study, we demonstrate that DnaJ binds a model non-native substrate with a low nanomolar dissociation constant and, more importantly, modulates the structure of its non-native state. The structural modulation achieved by DnaJ is different compared to that achieved by the DnaK–DnaJ complex. The nature of structural modulation exerted by DnaJ is suggestive of a unique unfolding activity on the non-native substrate by the chaperone. Furthermore, we demonstrate that the zinc binding motif along with the C-terminal substrate binding domain of DnaJ is necessary and sufficient for binding and the subsequent binding-induced structural alterations of the non-native substrate. We hypothesize that this hitherto unknown structural alteration of non-native states by DnaJ might be important for its chaperoning activity by removing kinetic traps of the folding intermediates

Surface exposed IVL clusters.

<p>Representative snapshot of the native (A) and the I₂ structure (B), displaying IVL residues constituting a part of GroES-like binding motifs. In comparison to the native structure, these clusters are mostly solvent-exposed.</p

Specificity of RSG-1.2 Peptide Binding to RRE-IIB RNA Element of HIV-1 over Rev Peptide Is Mainly Enthalpic in Origin

Rev is an essential HIV-1 regulatory protein which binds to the Rev responsive element (RRE) present within the env gene of HIV-1 RNA genome. This binding facilitates the transport of the RNA to the cytoplasm, which in turn triggers the switch between viral latency and active viral replication. Essential components of this complex have been localized to a minimal arginine rich Rev peptide and stem IIB region of RRE. A synthetic peptide known as RSG-1.2 binds with high binding affinity and specificity to the RRE-IIB than the Rev peptide, however the thermodynamic basis of this specificity has not yet been addressed. The present study aims to probe the thermodynamic origin of this specificity of RSG-1.2 over Rev Peptide for RRE-IIB. The temperature dependent melting studies show that RSG-1.2 binding stabilizes the RRE structure significantly (DT m = 4.3uC), in contrast to Rev binding. Interestingly the thermodynamic signatures of the binding have also been found to be different for both the peptides. At pH 7.5, RSG-1.2 binds RRE-IIB with a Ka = 16.260.6610 7 M 21 where enthalpic change DH = 213.960.1 kcal/mol is the main driving force with limited unfavorable contribution from entropic change TDS = 22.860.1 kcal/mol. A large part of DH may be due to specific stacking between U72 and Arg15. In contrast binding of Rev (K a = 3.160.4610 7 M 21) is driven mainly by entropy (DH = 0 kcal/mol and TDS = 10.260.2 kcal/mol) which arises fro

Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding

<div><p>Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The <i>E.coli</i> chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native <i>β</i>-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central “hubs”. Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates.</p></div

Conformational heterogeneity.

<p>A) Representative snapshots showing disruption of TIM barrel topology with <i>α</i> and <i>β</i>-region shown in blue and red color, respectively. B) Time evolution of distance between <i>α</i> and <i>β</i>-core for all three 400 K simulation shown in three shades of orange. For comparison, native 300 K is also shown in black. C) Time occurrence of representative <i>β</i>-core residues. The brown color represents the existence of beta-sheet secondary structure based on the dihedral angles.</p