Content
2010
Approaching the Secrets of N-Glycosylation in Aspergillus fumigatus: Characterization of the AfOch1 Protein
29.12.2010
The mannosyltransferase Och1 is the key enzyme for synthesis of elaborated protein N-glycans in yeast. In filamentous fungi genes implicated in outer chain formation are present, but their function is unclear. In this study we have analyzed the Och1 protein of Aspergillus fumigatus. We provide first evidence that poly-mannosylated N-glycans exist in A. fumigatus and that their synthesis requires AfOch1 activity. This implies that AfOch1 plays a similar role as S. cerevisiae ScOch1 in the initiation of an N-glycan outer chain. A Δafoch1 mutant showed normal growth under standard and various stress conditions including elevated temperature, cell wall and oxidative stress. However, sporulation of this mutant was dramatically reduced in the presence of high calcium concentrations, suggesting that certain proteins engaged in sporulation require N-glycan outer chains to be fully functional. A characteristic feature of AfOch1 and Och1 homologues from other filamentous fungi is a signal peptide that clearly distinguishes them from their yeast counterparts. However, this difference does not appear to have consequences for its localization in the Golgi. Replacing the signal peptide of AfOch1 by a membrane anchor had no impact on its ability to complement the sporulation defect of the Δafoch1 strain. The mutant triggered a normal cytokine response in infected murine macrophages, arguing against a role of outer chains as relevant Aspergillus pathogen associated molecular patterns. Infection experiments provided no evidence for attenuation in virulence; in fact, according to our data the Δafoch1 mutant may even be slightly more virulent than the control strains.
Mixed Hsp90–cochaperone complexes are important for the progression of the reaction cycle
19.12.2010
Nature Structural & Molecular Biology,
2011,
18,
61-6
published on 19.12.2010
Nature Structural & Molecular Biology, online article
Nature Structural & Molecular Biology, online article
The chaperone cycle of heat shock protein-90 (Hsp90) involves progression through defined complexes with different cochaperones. It is still enigmatic how the exchange of cochaperones is regulated. The first cochaperone entering the cycle is the Hsp90 ATPase inhibitor Sti1 (Hop in human), which later is replaced by a prolyl isomerase (PPIase) and p23. We found, unexpectedly, that one Sti1 molecule is sufficient to completely inhibit the ATPase of the Hsp90 dimer. Upon addition of a PPIase cochaperone to the Hsp90–Sti1 complex, an asymmetric ternary complex is preferentially formed. This PPIase–Hsp90–Sti1 intermediate is important for the progression of the cycle. To expel the bound Sti1, the concerted action of ATP and p23 is required. This mechanism, which is strictly conserved between the yeast and human Hsp90 systems, presents an example of how, in a cyclic process, directionality of assembly and disassembly of protein complexes can be achieved.
Ferredoxin:NADPH oxidoreductase is recruited to thylakoids by binding to a polyproline type II helix in a pH-dependent manner
09.11.2010
PNAS,
2010,
doi: 10.1073/pnas.1009124107,
vol. 107 no. 45 19260-19265
published on 09.11.2010
PNAS, online article
PNAS, online article
Ferredoxin:NADPH oxidoreductase (FNR) is a key enzyme of photosynthetic electron transport required for generation of reduction equivalents. Recently, two proteins were found to be involved in membrane-anchoring of FNR by specific interaction via a conserved Ser/Pro-rich motif: Tic62 and Trol. Our crystallographic study reveals that the FNR-binding motif, which forms a polyproline type II helix, induces self-assembly of two FNR monomers into a back-to-back dimer. Because binding occurs opposite to the FNR active sites, its activity is not affected by the interaction. Surface plasmon resonance analyses disclose a high affinity of FNR to the binding motif, which is strongly increased under acidic conditions. The pH of the chloroplast stroma changes dependent on the light conditions from neutral to slightly acidic in complete darkness or to alkaline at saturating light conditions. Recruiting of FNR to the thylakoids could therefore represent a regulatory mechanism to adapt FNR availability/activity to photosynthetic electron flow.
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The Heat Shock Response: Life on the Verge of Death
22.10.2010
Molecular Cell,
2010,
doi:10.1016/j.molcel.2010.10.006,
Volume 40, Issue 2, 253-266,
published on 22.10.2010
Molecular Cell, online article
Molecular Cell, online article
Organisms must survive a variety of stressful conditions, including sudden temperature increases that damage important cellular structures and interfere with essential functions. In response to heat stress, cells activate an ancient signaling pathway leading to the transient expression of heat shock or heat stress proteins (Hsps). Hsps exhibit sophisticated protection mechanisms, and the most conserved Hsps are molecular chaperones that prevent the formation of nonspecific protein aggregates and assist proteins in the acquisition of their native structures. In this Review, we summarize the concepts of the protective Hsp network.
Protein import into chloroplasts—How chaperones feature into the game
02.10.2010
Biochimica et Biophysica Acta,
2010,
doi:10.1016/j.bbamem.2010.07.021,
Volume 1808, Issue 3, Pages 901-911
published on 02.10.2010
Biochimica et Piophysica Acta, online article
Biochimica et Piophysica Acta, online article
Chloroplasts originated from an endosymbiotic event, in which an ancestral photosynthetic cyanobacterium was engulfed by a mitochondriate eukaryotic host cell. During evolution, the endosymbiont lost its autonomy by means of a massive transfer of genetic information from the prokaryotic genome to the host nucleus. Consequently, the development of protein import machineries became necessary for the relocation of proteins that are now nuclear-encoded and synthesized in the cytosol but destined for the chloroplast. Organelle biogenesis and maintenance requires a tight coordination of transcription, translation and protein import between the host cell and the organelle. This review focuses on the translocation complexes in the outer and inner envelope membrane with a special emphasis on the role of molecular chaperones. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.
Hsp12 Is an Intrinsically Unstructured Stress Protein that Folds upon Membrane Association and Modulates Membrane Function
27.08.2010
Hsp12 of S. cerevisiae is upregulated several 100-fold in response to stress. Our phenotypic analysis showed that this protein is important for survival of a variety of stress conditions, including high temperature. In the absence of Hsp12, we observed changes in cell morphology under stress conditions. Surprisingly, in the cell, Hsp12 exists both as a soluble cytosolic protein and associated to the plasma membrane. The in vitro analysis revealed that Hsp12, unlike all other Hsps studied so far, is completely unfolded; however, in the presence of certain lipids, it adopts a helical structure. The presence of Hsp12 does not alter the overall lipid composition of the plasma membrane but increases membrane stability.
Dynamics of heat shock protein 90 C-terminal dimerization is an important part of its conformational cycle
24.08.2010
The molecular chaperone heat shock protein 90 (Hsp90) is an important and abundant protein in eukaryotic cells, essential for the activation of a large set of signal transduction and regulatory proteins. During the functional cycle, the Hsp90 dimer performs large conformational rearrangements. The transient N-terminal dimerization of Hsp90 has been extensively investigated, under the assumption that the C-terminal interface is stably dimerized. Using a fluorescence-based single molecule assay and Hsp90 dimers caged in lipid vesicles, we were able to separately observe and kinetically analyze N- and C-terminal dimerizations. Surprisingly, the C-terminal dimer opens and closes with fast kinetics. The occupancy of the unexpected C-terminal open conformation can be modulated by nucleotides bound to the N-terminal domain and by N-terminal deletion mutations, clearly showing a communication between the two terminal domains. Moreover our findings suggest that the C- and N-terminal dimerizations are anticorrelated. This changes our view on the conformational cycle of Hsp90 and shows the interaction of two dimerization domains.
Structure of hibernating ribosomes studied by cryoelectron tomography in vitro and in situ
23.08.2010
JCB,
2010,
doi:10.1083/jcb.201005007,
Volume 190, Issue 4, Pages 613-621
published on 23.08.2010
JCB, online article
JCB, online article
Ribosomes arranged in pairs (100S) have been related with nutritional stress response and are believed to represent a “hibernation state.” Several proteins have been identified that are associated with 100S ribosomes but their spatial organization has hitherto not been characterized. We have used cryoelectron tomography to reveal the three-dimensional configuration of 100S ribosomes isolated from starved Escherichia coli cells and we have described their mode of interaction. In situ studies with intact E. coli cells allowed us to demonstrate that 100S ribosomes do exist in vivo and represent an easily reversible state of quiescence; they readily vanish when the growth medium is replenished.
A new concept for ferredoxin–NADP(H) oxidoreductase binding to plant thylakoids
18.08.2010
Trends in Plant Science,
2010,
doi:10.1016/j.tplants.2010.08.008,
Volume 15, Issue 11, Pages 608-613
published on 18.08.2010
Trends in Plant Science, online article
Trends in Plant Science, online article
During the evolution of photosynthesis, regulatory circuits were established that allow the precise coupling of light-driven electron transfer chains with downstream processes such as carbon fixation. The ferredoxin (Fd):ferredoxin–NADP+ oxidoreductase (FNR) couple is an important mediator for these processes because it provides the transition from exclusively membrane-bound light reactions to the mostly stromal metabolic pathways. Recent progress has allowed us to revisit how FNR is bound to thylakoids and to revaluate the current view that only membrane-bound FNR is active in photosynthetic reactions. We argue that the vast majority of thylakoid-bound FNR of higher plants is not necessary for photosynthesis. We furthermore propose that the correct distribution of FNR between stroma and thylakoids is used to efficiently regulate Fd-dependent electron partitioning in the chloroplast.
Redox extends its regulatory reach to chloroplast protein import
03.08.2010
Trends in Plant Science,
2010,
doi:10.1016/j.tplants.2010.06.002,
Volume 15, Issue 9, Pages 515-521
published on 03.08.2010
Trends in Plant Science, online article
Trends in Plant Science, online article
The import of chloroplast proteins synthesized in the cytosol of a plant cell is mediated by two multiprotein complexes or translocons located at the outer and inner membranes of the chloroplast envelope, respectively, TOC and TIC. These complexes integrate different signals to assure the timely transport of proteins into the chloroplast in accordance with the metabolic and developmental needs of the cell. The past few years have witnessed the emergence of redox as a regulator of the protein transport process. Here, we discuss evidence that the metabolic redox state of the chloroplast regulates the import of preproteins by altering either the activity or composition of participating transport components. It appears that, through these redox changes, chloroplasts communicate with other compartments of the plant cell.
Structural and Mechanical Hierarchies in the Alpha Crystallin Domain Dimer of the Hyperthermophilic Small Heat Shock Protein Hsp16.5
30.07.2010
Journal of Molecular Biology,
2010,
doi:10.1016/j.jmb.2010.05.065,
Volume 400, Issue 5, Pages 1046-1056
published on 30.07.2010
Journal of Molecular Biology, online article
Journal of Molecular Biology, online article
In biological systems, proteins rarely act as isolated monomers. Association to dimers or higher oligomers is a commonly observed phenomenon. As an example, small heat shock proteins form spherical homo-oligomers of mostly 24 subunits, with the dimeric αlpha-crystallin domain as the basic
structural unit. The structural hierarchy of this complex is key to its function as a molecular chaperone. In this article, we analyze the folding and association of the basic building block, the αlpha-crystallin domain dimer, from the hyperthermophilic archaeon Methanocaldococcus jannaschii Hsp16.5 in detail. Equilibrium denaturation experiments reveal that the αlpha-crystallin domain dimer is highly stable against chemical denaturation. In these experiments, protein dissociation and unfolding appear to follow an “all-ornone” mechanism with no intermediate monomeric species populated. When the mechanical stability was determined by single-molecule force
spectroscopy, we found that the αlpha-crystallin domain dimer resists high forces when pulled at its termini. In contrast to bulk denaturation, stable monomeric unfolding intermediates could be directly observed in the mechanical unfolding traces after the αlpha-crystallin domain dimer had been dissociated by force. Our results imply that for this hyperthermophilic
member of the small heat shock protein family, assembly of the spherical 24mer starts from folded monomers, which readily associate to the dimeric structure required for assembly of the higher oligomer.
NETs formed by human neutrophils inhibit growth of the pathogenic mold Aspergillus fumigatus
17.07.2010
Microbes and Infection,
2010,
doi:10.1016/j.micinf.2010.06.009,
Volume 12, Pages 928-936
published on 17.07.2010
Microbes and Infection, online article
Microbes and Infection, online article
Neutrophil extracellular traps (NETs) represent a distinct mechanism to control and eliminate microbial infections. Our results show that conidia and germ tubes of the human pathogenic mold Aspergillus fumigatus are able to trigger the formation of NETs. Viable fungal cells are not essentially required for this hostepathogen interaction. Neutrophils engulf conidia and thereby inhibit their germination, a process that is independent of NETosis. In the experimental set-up used in this study neutrophils do not kill germ tubes, but reduce their polar growth and this inhibition depends on NETs as it can be overcome by the addition of DNase-1. The Zn2þ chelator calprotectin is associated with the Aspergillus- induced NETs and addition of Zn2þ abrogates the NET-mediated growth inhibition. In summary, our data provide evidence that NETs are not sufficient to kill A. fumigatus, but might be a valuable tool to confine infection.
Chaperonin-Catalyzed Rescue of Kinetically Trapped States in Protein Folding
09.07.2010
Cell,
2010,
doi:10.1016/j.cell.2010.05.027,
Volume 142, Issue 1, 112-122
published on 09.07.2010
Cell, online article
Cell, online article
GroEL and GroES form a chaperonin nano-cage for single protein molecules to fold in isolation. The folding properties that render a protein chaperonin dependent are not yet understood. Here, we address this question using a double mutant of the maltosebinding protein DM-MBP as a substrate. Upon spontaneous refolding, DM-MBP populates a kinetically trapped intermediate that is collapsed but structurally disordered. Introducing two long-range disulfide bonds into DM-MBP reduces the entropic folding barrier of this intermediate and strongly accelerates native state formation. Strikingly, steric confinement
of the protein in the chaperonin cage mimics the kinetic effect of constraining disulfides on folding, in a manner mediated by negative charge clusters in the cage wall. These findings suggest that chaperonin dependence correlates with the tendency of
proteins to populate entropically stabilized folding intermediates. The capacity to rescue proteins from such folding traps may explain the uniquely essential role of chaperonin cages within the cellular chaperone network.
Dissecting the Alternatively Folded State of the Antibody Fab Fragment
25.06.2010
Journal of Molecular Biology,
2010,
doi:10.1016/j.jmb.2010.04.032,
Volume 399, Issue 5, Pages 719-730
published on 25.06.2010
Journal of Molecular Biology, online article
Journal of Molecular Biology, online article
Intact antibodies and antigen binding fragments (Fab) have been previously shown to form an alternatively folded state (AFS) at low pH. This state consists primarily of secondary structure interactions, with reduced tertiary structure content. The AFS can be distinguished from the molten globule
state by the formation of nonnative structure and, in particular, its high stability. In this study, the isolated domains of the MAK33 (murine monoclonal antibody of the subtype Kappa/IgG1) Fab fragment were investigated under conditions that have been reported to induce the AFS. Surprising differences in the ability of individual domains to form the AFS
were observed, despite the similarities in their native structures. All Fab domains were able to adopt the AFS, but only for Vh (variable domain of the heavy chain) could a significant amount of tertiary structure be detected
and different conditions were needed to induce the AFS. VH, the least stable of the domains under physiological conditions, was the most stable in the AFS, yet all domains showed significant stability against thermal and chemical unfolding in their AFS. Formation of the AFS was found to generally proceed via the unfolded state, with similar rates for most of the domains. Taken together, our data reveal striking differences in the biophysical properties of the AFS of individual antibody domains that reflect the variation possible for domains of highly homologous native structures. Furthermore, they allow individual domain contributions to be
dissected from specific oligomer effects in the AFS of the antibody Fab fragment.
The plant PRAT proteins – preprotein and amino acid transport in mitochondria and chloroplasts
21.06.2010
Plant Biology,
2010,
doi:10.1111/j.1438-8677.2010.00357.x,
Volume 12, pages 42–55
published on 21.06.2010
Plant Biology, online article
Plant Biology, online article
The membrane proteins of the plant preprotein and amino acid transporter (PRAT) superfamily all share common structural elements, such as four membrane-spanning α-helices. Interestingly they display diverse localisation to outer and inner membranes of chloroplasts and mitochondria. Furthermore, they fulfil different functions in preprotein translocation as well as amino acid transport across these membranes. This review summarises current knowledge on precursor protein import and amino acid transport in plastids and mitochondria and provides an overview of the distinct tasks and features of members of the PRAT superfamily in the model plant Arabidopsis thaliana.
Intramembrane Proteolysis of Mgm1 by the Mitochondrial Rhomboid Protease Is Highly Promiscuous Regarding the Sequence of the Cleaved Hydrophobic Segment
15.06.2010
Journal of Molecular Biology,
2010,
doi:10.1016/j.jmb.2010.06.014,
Volume 401, Issue 2, Pages 182-193
published on 15.06.2010
JMB, online article
JMB, online article
Rhomboids are a family of intramembrane serine proteases that are conserved in bacteria, archaea, and eukaryotes. They are required for numerous fundamental cellular functions such as quorum sensing, cell signaling, and mitochondrial dynamics. Mitochondrial rhomboids form an evolutionarily distinct class of rhomboids. It is largely unclear how their activity is controlled and which substrate determinants are responsible for recognition and cleavage. We investigated these requirements for the mitochondrial rhomboid protease Pcp1 and its substrate Mgm1. In contrast to several other rhomboid proteases, Pcp1 does not require helix-breaking amino acids in the cleaved hydrophobic region of Mgm1, termed 'rhomboid cleavage region' (RCR). Even transmembrane segments of inner membrane proteins that are normally not processed by Pcp1 become cleavable when put in place of the authentic RCR of Mgm1. We further show that mutational alterations of a highly negatively charged region located C-terminally to the RCR led to a strong processing defect. Moreover, we show that the determinants required for Mgm1 processing by mitochondrial rhomboid protease are conserved during evolution, as PARL (the human ortholog of Pcp1) showed similar substrate requirements. These results suggest a surprising promiscuity of the mitochondrial rhomboid protease regarding the sequence requirements of the cleaved hydrophobic segment. We propose a working hypothesis on how the mitochondrial rhomboid protease can, despite this promiscuity, achieve a high specificity in recognizing Mgm1. This hypothesis relates to the exceptional biogenesis pathway of Mgm1.
Independent evolution of the core domain and its flanking sequences in small heat shock proteins
25.05.2010
The FASEB Journal,
2010,
doi: 10.1096/fj.10-156992,
vol. 24 no. 10 3633-3642
published on 25.05.2010
The FASEB Journal, online article
The FASEB Journal, online article
Small heat shock proteins (sHsps) are molecular chaperones involved in maintaining protein homeostasis; they have also been implicated in protein folding diseases and in cancer. In this protein family, a conserved core domain, the so-called alpha-crystallin or Hsp20 domain, is flanked by highly variable, nonconserved sequences that are essential for chaperone function. Analysis of 8714 sHsps revealed a broad variation
of primary sequences within the superfamily as well as phyla-dependent differences. Significant variations were found in the number of sHsps per genome, their amino acid composition, and the length distribution of the different sequence parts. Reconstruction of the evolutionary tree for the sHsp superfamily shows that the flanking regions fall into several subgroups, indicating that they were remodeled several times in parallel but independent of the evolution of the alpha-crystallin domain. The evolutionary history of sHsps is thus set apart from that of other protein families in that two exon boundaryindependent strategies are combined: the evolution of
the conserved alpha-crystallin domain and the independent evolution of the N- and C-terminal sequences. This scenario allows for increased variability in specific small parts of the protein and thus promotes functional and structural differentiation of sHsps, which is not reflected in the general evolutionary tree of species.
Farnesol misplaces tip-localized Rho proteins and inhibits cell wall integrity signalling in Aspergillus fumigatus
11.05.2010
Molecular Microbiology,
2010,
doi:10.1111/j.1365-2958.2010.07170.x,
Volume 76, Issue 5, Pages 1191-1204
published on 11.05.2010
Molecular Microbiology, online article
Molecular Microbiology, online article
Farnesol is known for inducing apoptosis in some fungi and mammalian cells. To evaluate its potential role as an antifungal agent, we studied its impact on the human pathogen Aspergillus fumigatus. We found that growth of A. fumigatus wild type is inhib- ited, but two cell wall mutants, Dmnt1 and DglfA, are much more susceptible to farnesol. This susceptibil- ity is partially rescued by osmotic stabilization, sug- gesting that farnesol is a cell wall perturbing agent. However, farnesol does not activate but inhibit the cell wall integrity (CWI) pathway. Remarkably, mutants lacking AfMkk2 or AfMpkA, two kinases essential for CWI signalling, are also highly suscep- tible to farnesol, suggesting that its mode of action goes beyond inhibition of CWI signalling. Farnesyl derivatives are known for interfering with the function of prenylated proteins. We analysed the subcellular localization of two prenylated Rho family GTPases, AfRho1 and AfRho3, which are implicated in control- ling CWI and the cytoskeleton. We found that under normal growth conditions AfRho1 and AfRho3 pre- dominantly localize to the hyphal tip. After farnesol treatment this localization is rapidly lost, which is accompanied by swelling of the hyphal tips. Parallel displacement of tropomyosin from the tips suggests a concomitant disorganization of the apical actin cytoskeleton.
AfMkk2 is required for cell wall integrity signaling, adhesion, and full virulence of the human pathogen Aspergillus fumigatus
07.05.2010
International Journal of Medical Microbiology,
2010,
doi:10.1016/j.ijmm.2010.03.001,
Volume 300, Issue 7, Pages 496-502
published on 07.05.2010
International Journal of Medical Microbiology, online article
International Journal of Medical Microbiology, online article
The cell wall integrity (CWI) pathway, best characterized in S. cerevisiae, is strikingly conserved in Aspergillus species. We analyzed the importance of AfMkk2, a CWI signaling kinase, for virulence and antifungal therapy in the human pathogen A. fumigatus. A mutant lacking AfMkk2 is less adherent to glass and plastic surfaces and shows increased sensitivity to alkaline pH stress and antifungals. Rather than AfMpkA, the target kinase of AfMkk2, AfMpkB is activated in the mutant under cell wall stress. Interestingly, the mutant lacking AfMkk2 shows an enhanced sensitivity to posaconazole and voriconazole. And in agreement with its sensitivity to moderate temperatures, it is less virulent in a murine infection model. Our data underline the importance of mkk2 for the fitness, but also for the pathogenicity of A. fumigatus.
Evolution of Escherichia coli for growth at high temperatures
20.04.2010
The Journal of Biological Chemistry,
2010,
doi: 10.1074/jbc.M110.103374,
pp. 19029–19034,
published on 20.04.2010
The Journal of Biological Chemistry, online article
The Journal of Biological Chemistry, online article
Evolution depends on the acquisition of genomic mutations that increase cellular fitness. Here, we evolved Escherichia coli MG1655 cells to grow at extreme temperatures. We obtained a maximum growth temperature of 48.5°C, which was not further increased upon continuous cultivation at this temperature for over 600 generations. Despite a permanently induced heat shock response in thermoresistant cells, only exquisitely high GroEL/GroES levels are essential for growth at 48.5°C. They depend on the presence of lysyl-tRNA-synthetase, LysU, because deletion of lysU rendered thermoresistant cells thermosensitive. Our data suggest that GroEL/GroES are especially required for the folding of mutated proteins generated during evolution. GroEL/GroES therefore appear as mediators of evolution of extremely heat-resistant E. coli cells.
The Conformational Dynamics of the Mitochondrial Hsp70 Chaperone
09.04.2010
Molecular Cell,
2010,
doi:10.1016/j.molcel.2010.03.010,
Volume 38, Issue 1, Pages 89-100
published on 09.04.2010
Molecular Cell, online article
Molecular Cell, online article
Heat shock proteins 70 (Hsp70) represent a ubiquitous and conserved family of molecular chaperones involved in a plethora of cellular processes. The dynamics of their ATP hydrolysis-driven and cochaperone-regulated conformational cycle are poorly understood. We used fluorescence spectroscopy to analyze, in real time and at single-molecule resolution, the effects of nucleotides and cochaperones on the conformation of Ssc1, a mitochondrial member of the family. We report that the conformation of its ADP state is unexpectedly heterogeneous, in contrast to a uniform ATP state. Substrates are actively involved in determining the conformation of Ssc1. The J protein Mdj1 does not interact transiently with the chaperone, as generally believed, but rather is released slowly upon ATP hydrolysis. Analysis of the major bacterial Hsp70 revealed important differences between highly homologous members of the family, possibly explaining tuning of Hsp70 chaperones to meet specific functions in different organisms and cellular compartments.
An unlocking/relocking barrier in conformational fluctuations of villin headpiece subdomain
01.03.2010
A reversible structural unlocking reaction, in which the close-packed van der Waals interactions break cooperatively, has been found for the villin headpiece subdomain (HP35) using triplet-triplet-energy transfer to monitor conformational fluctuations from equilibrium. Unlocking is associated with an unfavorable enthalpy change (ΔH0 = 35 ± 4 kJ/mol) which is nearly compensated in free energy by the entropy change (ΔS0 = 112 ± 20 J·mol-1·K-1). The unlocking reaction has a time constant of about 1 μs at 5 °C and is enthalpy-limited with an activation energy of 32 ± 1 kJ/mol and a large Arrhenius preexponential factor of A = 7.5 × 1011 s-1. In the unlocked state a fast local conformational fluctuation with a time constant of 170 ns and a low activation barrier of 17 ± 1 kJ/mol leads to unfolding of the C-terminal helix and to its undocking from the core. On a much slower time scale, global unfolding occurs from the unlocked state. These results suggest that native protein structures are locked into conformations with low amplitude motions. Large scale motions and global unfolding require an initial structural unlocking step leading to a state with properties of a dry molten globule. The experiments additionally yielded information on the dynamics of loop formation between different positions in unfolded HP35. Comparison of the results with dynamics in unstructured model peptides indicates slightly decelerated kinetics of local loop formation in the C-terminal region which points at residual, nonrandom structure. Dynamics of long-range loop formation, in contrast, are not influenced by residual structure, which argues against unfolded state properties as molecular origin for ultrafast folding of HP35.
Regions Outside the Alpha-Crystallin Domain of the Small Heat Shock Protein Hsp26 Are Required for Its Dimerization
18.02.2010
Journal of Molecular Biology,
2010,
doi:10.1016/j.jmb.2010.02.022,
Volume 398, Issue 1, Pages 122-131
published on 18.02.2010
Journal of Molecular Biology, online article
Journal of Molecular Biology, online article
Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones. They form homo-oligomers, composed of mostly 24 subunits. The immunoglobulin-like α-crystallin domain, which is flanked by N- and C-terminal extensions, is the most conserved element in sHsps. It is
assumed to be the dimeric building block from which the sHsp oligomers are assembled. Hsp26 from Saccharomyces cerevisiae is a well-characterized member of this family.With a view to study the structural stability and oligomerization
properties of its alpha-crystallin domain, we produced a series of alpha-crystallin domain constructs. We show that a minimal α-crystallin domain can, against common belief, be monomeric and stably folded. Elongating either the N- or the C-terminus of this minimal αlpha-crystallin domain with the
authentic extensions leads to the formation of dimeric species. In the case of N-terminal extensions, their population is dependent on the presence of the complete so-called Hsp26 “middle domain”. For the C-terminal extensions, the presence of the conserved IXI motif of sHsps is necessary and sufficient
to induce dimerization, which can be inhibited by increasing ionic strength. Dimerization does not induce major changes in secondary structure of the Hsp26 αlpha-crystallin domain. A thermodynamic analysis of the monomeric and dimeric constructs revealed that dimers are not significantly stabilized
against thermal and chemical denaturation in comparison to monomers, supporting our notion that dimerization is not a prerequisite for the formation of a well-folded Hsp26 αlpha-crystallin domain.
Asymmetric Activation of the Hsp90 Dimer by Its Cochaperone Aha1
12.02.2010
Mol. Cell,
2010,
37(3),
344-354, doi:10.1016/j.molcel.2010.01.006
published on 12.02.2010
Molecular Cell, online article
Molecular Cell, online article
The chaperone Hsp90 is an ATP-dependent, dimeric molecular machine regulated by several cochaperones, including inhibitors and the unique ATPase activator Aha1. Here, we analyzed the mechanism of the Aha1-mediated acceleration of Hsp90 ATPase activity and identified the interaction surfaces of both proteins using multidimensional NMR techniques. For maximum activation of Hsp90, the two domains of Aha1 bind to sites in the middle and N-terminal domains of Hsp90 in a sequential manner. This binding induces the kinetically unfavored N terminally dimerized state of Hsp90, which primes for the hydrolysis-competent conformation. Surprisingly, this activation mechanism is asymmetric. The presence of one Aha1 molecule per Hsp90 dimer is sufficient to bridge the two subunits and to fully stimulate Hsp90 ATPase activity. This seems to functionalize the two subunits of the Hsp90 dimer in different ways, in that one subunit can be used for conformational ATPase regulation and the other for substrate protein processing.
Redox-regulation of protein import into chloroplasts and mitochondria: Similarities and differences.
01.02.2010
Plant Signal. Behav.,
2010,
5(2),
published on 01.02.2010
Plant Signaling and Behavior, online article
Plant Signaling and Behavior, online article
Redox signals play important roles in many developmental and metabolic processes, in particular in chloroplasts and mitochondria. Furthermore, redox reactions are crucial for protein folding via the formation of inter- or intramolecular disulfide bridges. Recently, redox signals were described to be additionally involved in regulation of protein import: in mitochondria, a disulfide relay system mediates retention of cystein-rich proteins in the intermembrane space by oxidizing them. Two essential proteins, the redox-activated receptor Mia40 and the sulfhydryl oxidase Erv1 participate in this pathway. In chloroplasts, it becomes apparent that protein import is affected by redox signals on both the outer and inner envelope: at the level of the Toc complex (translocon at the outer envelope of chloroplasts), the formation/reduction of disulfide bridges between the Toc components has a strong influence on import yield. Moreover, the stromal metabolic redox state seems to be sensed by the Tic complex (translocon at the inner envelope of chloroplasts) that is able to adjust translocation efficiency of a subgroup of redox-related preproteins accordingly. This review summarizes the current knowledge of these redox-regulatory pathways and focuses on similarities and differences between chloroplasts and mitochondria.
The many faces of the mitochondrial TIM23 complex
28.01.2010
Biochimica et Biophysica Acta,
2010,
doi:10.1016/j.bbabio.2010.01.026,
Volume 1797, Issues 6-7, Pages 1045-1054
published on 28.01.2010
Biochimica et Biophysica Acta, online article
Biochimica et Biophysica Acta, online article
The TIM23 complex in the inner membrane of mitochondria mediates import of essentially all matrix proteins and a large number of inner membrane proteins. Here we present an overview on the latest insights into the structure and function of this remarkable molecular machine.
Protein import into chloroplasts: The Tic complex and its regulation
25.01.2010
Biochimica et Biophysica Acta,
2010,
doi:10.1016/j.bbamcr.2010.01.015,
Volume 1803, Issue 6, Pages 740-747
published on 25.01.2010
Biochimica et Piophysica Acta, online article
Biochimica et Piophysica Acta, online article
Chloroplasts originated from an endosymbiotic event, in which an ancestral photosynthetic cyanobacterium was engulfed by a mitochondriate eukaryotic host cell. During evolution, the endosymbiont lost its autonomy by means of a massive transfer of genetic information from the prokaryotic genome to the host nucleus. Consequently, the development of protein import machineries became necessary for the relocation of proteins that are now nuclear-encoded and synthesized in the cytosol but destined for the chloroplast. Organelle biogenesis and maintenance requires a tight coordination of transcription, translation and protein import between the host cell and the organelle. This review focuses on the translocation complexes in the outer and inner envelope membrane with a special emphasis on the role of molecular chaperones. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.
Coupled chaperone action in folding and assembly of hexadecameric Rubisco
14.01.2010
Nature,
2010,
doi:10.1038/nature08651,
Volume 463, 197-202
published on 14.01.2010
Nature, online article
Nature, online article
Form I Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), a complex of eight large (RbcL) and eight small (RbcS) subunits, catalyses the fixation of atmospheric CO2 in photosynthesis. The limited catalytic efficiency of Rubisco has sparked extensive efforts to re-engineer the enzyme with the goal of enhancing agricultural productivity. To facilitate such efforts we analysed the formation of cyanobacterial form I Rubisco by in vitro reconstitution and cryo-electron microscopy. We show that RbcL subunit folding by the GroEL/GroES chaperonin is tightly coupled with assembly mediated by the chaperone RbcX2. RbcL monomers remain partially unstable and retain high affinity for GroEL until captured by RbcX2. As revealed by the structure of a RbcL8–(RbcX2)8 assembly intermediate, RbcX2 acts as a molecular staple in stabilizing the RbcL subunits as dimers and facilitates RbcL8 core assembly. Finally, addition of RbcS results in RbcX2 release and holoenzyme formation. Specific assembly chaperones may be required more generally in the formation of complex oligomeric structures when folding is closely coupled to assembly.
A coronin7 homolog with functions in actin-driven processes
12.01.2010
J. Biol. Chem.,
in press, doi:10.1074/jbc.M109.083725
published on 12.01.2010
J. Biol. Chem., online article
J. Biol. Chem., online article
Dictyostelium discoideum Coronin7 (DdCRN7) together with human Coronin7 (CRN7) and Pod-1 of Drosophila melanogaster and Caenorhabditis elegans belong to the coronin family of WD repeat domain containing proteins. Coronin7 proteins are characterized by two WD repeat domains that presumably fold into two beta-propeller structures. DdCRN7 shares highest homology with human CRN7, a protein with roles in membrane trafficking. DdCRN7 is present in the cytosol and accumulates in cell surface projections during movement and phago- and pinocytosis. Cells lacking CRN7 have altered chemotaxis and phagocytosis. Furthermore, loss of CRN7 affects the infection process by the pathogen Legionella pneumophila and allows a more efficient internalization of bacteria. To provide a mechanism for CNR7 action we studied actin related aspects. We could show that CRN7 binds directly to F-actin and protects actin filaments from depolymerization. CRN7 also associated with F-actin in vivo. It was present in the Triton X-100 insoluble cytoskeleton, colocalized with F-actin and its distribution was sensitive to drugs affecting the actin cytoskeleton. We propose that CRN7's role in chemotaxis and phagocytosis is through its effect on the actin cytoskeleton.
Role of Tim50 in the Transfer of Precursor Proteins from the Outer to the Inner Membrane of Mitochondria
01.03.2009
Molecular Biology of the Cell,
2009,
20,
1400 - 07
published on 01.03.2009
Molecular Biology of the Cell., online article
Molecular Biology of the Cell., online article
Transport of essentially all matrix and a number of inner membrane proteins is governed, entirely or in part, by
N-terminal presequences and requires a coordinated action of the translocases of outer and inner mitochondrial membranes
(TOM and TIM23 complexes). Here, we have analyzed Tim50, a subunit of the TIM23 complex that is implicated
in transfer of precursors from TOM to TIM23. Tim50 is recruited to the TIM23 complex via Tim23 in an interaction that
is essentially independent of the rest of the translocase. We find Tim50 in close proximity to the intermembrane space side
of the TOM complex where it recognizes both types of TIM23 substrates, those that are to be transported into the matrix
and those destined to the inner membrane, suggesting that Tim50 recognizes presequences. This function of Tim50
depends on its association with TIM23. We conclude that the efficient transfer of precursors between TOM and TIM23
complexes requires the concerted action of Tim50 with Tim23.
