Search

SummaryEnvironmental stress can result in substantial damage to proteins, membranes, and genetic material, impacting organismal survival1-3. Stress tolerance can be conferred by intrinsically disordered proteins (IDPs)4 that lack stable tertiary structure. IDPs from the large family of late embryogenesis abundant (LEA) proteins confer a fitness advantage when heterologously expressed5,6. Such protection suggests a general molecular function leading to stress tolerance, although the mechanisms remain unclear. Here, we report that a tardigrade LEA protein that confers stress tolerance in yeast acts as a molecular chaperone for the mitochondrial membrane. This protein, named HeLEA1, localizes to the mitochondrial matrix, and harbors conserved LEA sequence motifs that undergo dynamic disorder-to-helical transition upon binding to negatively charged membranes. Yeast expressing HeLEA1 show increased mitochondrial membrane fluidity, increased membrane potential, and enhanced tolerance to hyperosmotic stress under non-fermentative growth without significantly altering mitochondrial lipid composition or triggering a generic stress response. We demonstrate that membrane binding ameliorates excess surface tension, possibly by stabilizing lipid packing defects. Evolutionary analysis suggests that HeLEA1 homologs localize to different membrane-bound organelles and share similar sequence and biophysical features. We suggest that membrane chaperoning by LEA proteins represents a general biophysical solution that can operate across the domains of life.

Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.

Cardiolipin mediates dynamic receptor-channel interactions within the mitochondrial TIM23 protein import complex., The phospholipid cardiolipin mediates the functional interactions of proteins that reside within energy-conserving biological membranes. However, the molecular basis by which this lipid performs this essential cellular role is not well understood. We address this role of cardiolipin using the multisubunit mitochondrial TIM23 protein transport complex as a model system. The early stages of protein import by this complex require specific interactions between the polypeptide substrate receptor, Tim50, and the membrane-bound channel-forming subunit, Tim23. Using analyses performed in vivo, in isolated mitochondria, and in reductionist nanoscale model membrane systems, we show that the soluble receptor domain of Tim50 interacts with membranes and with specific sites on the Tim23 channel in a manner that is directly modulated by cardiolipin. To obtain structural insights into the nature of these interactions, we obtained the first small-angle x-ray scattering-based structure of the soluble Tim50 receptor in its entirety. Using these structural insights, molecular dynamics simulations combined with a range of biophysical measurements confirmed the role of cardiolipin in driving the association of the Tim50 receptor with lipid bilayers with concomitant structural changes, highlighting the role of key structural elements in mediating this interaction. Together, these results show that cardiolipin is required to mediate specific receptor-channel associations in the TIM23 complex. Our results support a new working model for the dynamic structural changes that occur within the complex during transport. More broadly, this work strongly advances our understanding of how cardiolipin mediates interactions among membrane-associated proteins.

The isolation and characterization of mitochondrial membrane proteins is technically challenging because they natively reside within the specialized environment of the lipid bilayer, an environment that must be recapitulated to some degree during reconstitution to ensure proper folding, stability, and function. Here we describe protocols for the assembly of a membrane protein into lipid bilayer nanodiscs in a series of cell-free reactions. Cell-free expression of membrane proteins circumvents problems attendant with in vivo expression such as cytotoxicity, low expression levels, and the formation of inclusion bodies. Nanodiscs are artificial membrane systems comprised of discoidal lipid bilayer particles bound by annuli of amphipathic scaffold protein that shield lipid acyl chains from water. They are therefore excellent platforms for membrane protein reconstitution and downstream solution-based biochemical and biophysical analysis. This chapter details the procedures for the reconstitution of a mitochondrial membrane protein into nanodiscs using two different types of approaches: cotranslational and posttranslational assembly. These strategies are broadly applicable for different mitochondrial membrane proteins. They are also applicable for the use of nanodiscs with distinct lipid compositions that are biomimetic for different mitochondrial membranes and that recapitulate lipid profiles associated with pathological disorders in lipid metabolism.

Amphipathic polymers have been widely used to maintain the solubility of membrane proteins and complexes following detergent solubilization. However, their ability to extract proteins directly from lipid bilayers has remained largely unexplored. Here we show that a copolymer composed of styrene and maleic acid pendant groups (SMA) extracts proteins form native membranes and reconstitutes them into polymer-bound lipoprotein particles. First, we found that the SMA copolymer disrupted the membranes of intact mitochondria in a concentration-dependent and saturable manner. This was evidenced by the collapse of the transmembrane electric field of the inner mitochondrial membrane and by the solubilization of mitochondrial membrane proteins, both of which were mediated by the SMA copolymer in a manner similar to that mediated by nonionic detergents. Second, following incubation of the SMA copolymer with mitochondrial membranes and chromatographic separation, we observed by transmission electron microscopy that the resulting polymer-bound particles were a monodisperse population of discoids. The dimensions of these particles were similar to those previously reported for particles derived from liposomes or proteoliposomes of synthetic phospholipids (Lipodisqs®). Finally, using mitochondrial respiratory Complex IV (cytochrome c oxidase) as a model enzyme, we demonstrate that the SMA copolymer can extract even large, multicomponent complexes from native lipid bilayers and maintain them in a fully functional state amenable to solution-based biophysical studies. This novel approach to membrane protein reconstitution obviates the requirement for detergents and is therefore better suited to preserving native annular lipids and protein stability in comparison with traditional solubilization techniques.

Nine of ten methylated nucleotides of Escherichia coli 16 S rRNA are conserved in Mycobacterium tuberculosis. All the 10 different methyltransferases are known in E. coli, whereas only TlyA and GidB have been identified in mycobacteria. Here we have identified Rv2966c of M. tuberculosis as an ortholog of RsmD protein of E. coli. We have shown that rv2966c can complement rsmD-deleted E. coli cells. Recombinant Rv2966c can use 30 S ribosomes purified from rsmD-deleted E. coli as substrate and methylate G966 of 16 S rRNA in vitro. Structure determination of the protein shows the protein to be a two-domain structure with a short hairpin domain at the N terminus and a C-terminal domain with the S-adenosylmethionine-MT-fold. We show that the N-terminal hairpin is a minimalist functional domain that helps Rv2966c in target recognition. Deletion of the N-terminal domain prevents binding to nucleic acid substrates, and the truncated protein fails to carry out the m(2)G966 methylation on 16 S rRNA. The N-terminal domain also binds DNA efficiently, a property that may be utilized under specific conditions of cellular growth.

Objective—To evaluate the mechanical properties of canine carpal ligaments for use in a finite element model of the canine antebrachium. Sample Population—26 forelimbs obtained from cadavers of 13 dogs euthanized for reasons unrelated to this study. Procedures—6 ligaments (medial collateral, lateral collateral, palmar ulnocarpal, palmar radiocarpal, accessorometacarpal-V, and accessorometacarpal-IV) were evaluated. Quasistatic tensile tests were performed on all specimens (n = 8 specimens/ligament) by use of a servohydraulic materials testing system in conjunction with a 6-df load cell. Each specimen was preconditioned for 10 cycles by applying 2% strain by use of a Haversine waveform. Tension was subsequently applied to each specimen at a strain rate of 0.5%/s until ligament failure. Results—Significant differences in modulus of elasticity were detected among the ligaments. Elastic modulus did not differ significantly between the 2 accessorometacapal ligaments, between the 2 collateral ligaments, or between the 2 palmar carpal ligaments. Ligaments were classified into 3 groups (accessorometacarpal ligaments, intra-articular ligaments, and palmar carpal ligaments), and significant differences were detected among the 3 ligament groups. The accessorometacarpal ligaments had a relatively high elastic modulus, compared with results for the other ligaments. The medial and lateral collateral ligaments had the lowest elastic modulus of any of the ligaments tested. Conclusions and Clinical Relevance—These results indicated a strong function-elastic modulus relationship for the 6 ligaments tested. The mechanical properties described here will be of use in creating a finite element model of the canine antebrachium.

In the active HER receptor dimers, kinases play distinct roles; one is the catalytically active kinase and the other is its allosteric activator. This specialization enables signaling by the catalytically inactive HER3, which functions exclusively as an allosteric activator upon heterodimerization with other HER receptors. It is unclear whether the allosteric activation mechanism evolved before HER receptors functionally specialized. We determined the crystal structure of the kinase domain of the only EGF receptor in Caenorhabditis elegans, LET-23. Our structure of a non-human EGFR kinase reveals autoinhibitory features conserved in the human counterpart. Strikingly, mutations within the putative allosteric dimer interface abrogate activity of the isolated LET-23 kinase and of the full-length receptor despite these regions being only partially conserved with human EGFR. Our results indicate that ancestral EGFRs have built-in features that poise them for allosteric activation that could facilitate emergence of the catalytically dead, yet functional, orthologs.

Background The reconstitution of membrane proteins and complexes into nanoscale lipid bilayer structures has contributed significantly to biochemical and biophysical analyses. Current methods for performing such reconstitutions entail an initial detergent-mediated step to solubilize and isolate membrane proteins. Exposure to detergents, however, can destabilize many membrane proteins and result in a loss of function. Amphipathic copolymers have recently been used to stabilize membrane proteins and complexes following suitable detergent extraction. However, the ability of these copolymers to extract proteins directly from native lipid bilayers for subsequent reconstitution and characterization has not been explored. Results The styrene-maleic acid (SMA) copolymer effectively solubilized membranes of isolated mitochondria and extracted protein complexes. Membrane complexes were reconstituted into polymer-bound nanoscale discs along with endogenous lipids. Using respiratory Complex IV as a model, these particles were shown to maintain the enzymatic activity of multicomponent electron transporting complexes. Conclusions We report a novel process for reconstituting fully operational protein complexes directly from cellular membranes into nanoscale lipid bilayers using the SMA copolymer. This facile, single-step strategy obviates the requirement for detergents and yields membrane complexes suitable for structural and functional studies.

Tim23, the central subunit of the TIM23 protein-translocation complex, forms a voltage-gated channel in the mitochondrial inner membrane (MIM), an energy-conserving membrane that generates a proton-motive force to drive vital processes. Using high-resolution fluorescence mapping of a channel-facing transmembrane segment (TMS2) of Tim23 from Saccharomyces cerevisiae, we demonstrate that changes in the energized state of the MIM cause marked structural alterations in the channel region. In an energized membrane, TMS2 forms a continuous α-helix that is inaccessible to the aqueous intermembrane space (IMS). Upon depolarization, the helical periodicity of TMS2 is disrupted, and the channel becomes exposed to the IMS. Kinetic measurements confirm that changes in TMS2 conformation coincide with depolarization. These results reveal how the energized state of the membrane drives functionally relevant structural dynamics in membrane proteins coupled to processes such as channel gating.

The TIM23 complex of the mitochondrial inner membrane mediates the translocation and integration of nuclear-encoded polypeptides that are targeted to the mitochondrion. Using a host of in organello and model membrane systems, we have analyzed the conformational alterations of key TIM23 subunits and site-specific interactions that occur between subunits during different steps of the transport process. First, using a fluorescence mapping strategy, we have analyzed the structural dynamics of the central voltage-gated channel-forming subunit, Tim23. We show dramatic alterations in the conformation of Tim23 transmembrane segments of the channel region that occur in a manner coupled to the magnitude of the proton-motive force across the inner membrane. Second, using a series of model membrane systems and reductionist approaches, we have analyzed site-specific interactions that occur between the Tim23 channel and the central receptor Tim50. Using Tim23 reconstituted into nanodiscs of different lipid composition, we identify the Tim50-interactive regions within the soluble and channel domains of Tim23. We find a critical role for cardiolipin, the signature anionic phospholipid of the mitochondrion, in promoting Tim23-Tim50 interactions. Using a combination of biochemical, mass spectrometry, and molecular dynamics approaches, we identify sites of the Tim50 receptor that bind to cardiolipin-containing lipid bilayers. The headgroup and acyl chain regions of cardiolipin that are critical in mediating Tim50 binding have been addressed using mitochondria from yeast strains defective in different steps of cardiolipin remodeling as well as model membranes of different lipid composition. Finally, we use small angle x-ray scattering (SAXS) to generate ab initio models of the Tim50 receptor domain and its lipid-interactive surface. These results illuminate the early receptor-mediated stages of protein translocation by the TIM23 complex and on the role of cardiolipin in promoting subunit interactions within the translocon.

The TIM23 Complex of the mitochondrial inner membrane (IM) is a multi-component assembly that mediates the translocation of matrix-targeted precursor proteins as well as the integration of membrane proteins. This complex is energetically coupled to both an ATPase motor and the electrochemical proton potential across the IM. The central subunit of this complex, Tim23, forms a voltage-gated channel and contains a large soluble domain in the intermembrane space that functions as a substrate receptor. To analyze the structural dynamics that are attendant with changes in the energized state of the membrane, we employed a high-resolution fluorescence mapping approach in which small extrinsic probes were cotranslationally incorporated into specific sites of Tim23 from Saccharomyces cerevisiae and analyzed in active mitochondria by steady-state and time-resolved measurements under different physiologically relevant states. Analysis of the channel-facing transmembrane segment (TMS2) of Tim23 revealed that changes in the energized status of the inner membrane caused dramatic structural alterations in the channel region. In an energized membrane, TMS2 formed a continuous α-helix that was inaccessible to the aqueous intermembrane space. Upon depolarization, the helical periodicity of TMS2 was disrupted and the channel became exposed to the IMS. Real time kinetic measurements confirmed that changes in TMS2 conformation coincided with depolarization. This analysis is extended to the soluble receptor domain of Tim23, where we show proton-motive force-coupled structural changes and key protein interactions that are mediated by specific lipids within the inner membrane. These results reveal how the energized state of the membrane drives functionally relevant structural dynamics in membrane proteins that are coupled to processes such as channel gating.