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We have identified a peptide substrate for molecular chaperone Hsp40 Ydj1 by utilizing the combination of phage display library screening and isothemol titration calirimetry (ITC). The initial peptide substrate screening for Hsp40 Ydj1 has been carried out by utilizing a 7-mer phage display library. The peptide sequences from the bio-panning were synthesized and object to the direct affinity measurement for Hsp40 Ydj1 by isothemol titration calirimetry studies. The peptide which has the measurable affinity with Ydj1 shows enriched hydrophobic residues in the middle of the substrate fragment. The peptide substrate specificity for molecular chaperone Hsp40 has been analyzed.

In this study, we presented an AISID method extending AlphaFold-Multimer's success in structure prediction towards identifying specific protein interactions with an optimized AISIDscore. The method was tested to identify the binding proteins in 18 human TNFSF (Tumor Necrosis Factor superfamily) members for each of 27 human TNFRSF (TNF receptor superfamily) members. For each TNFRSF member, we ranked the AISIDscore among the 18 TNFSF members. The correct pairing resulted in the highest AISIDscore for 13 out of 24 TNFRSF members which have known interactions with TNFSF members. Out of the 33 correct pairing between TNFSF and TNFRSF members, 28 pairs could be found in the top five (including 25 pairs in the top three) seats in the AISIDscore ranking. Surprisingly, the specific interactions between TNFSF10 (TNF-related apoptosis-inducing ligand, TRAIL) and its decoy receptors DcR1 and DcR2 gave the highest AISIDscore in the list, while the structures of DcR1 and DcR2 are unknown. The data strongly suggests that AlphaFold-Multimer might be a useful computational screening tool to find novel specific protein bindings. This AISID method may have broad applications in protein biochemistry, extending the application of AlphaFold far beyond structure predictions. [ABSTRACT FROM AUTHOR]

The noradrenergic link: Norepinephrine (NE) and the noradrenergic system play a main role in cognition and noradrenergic changes have been documented in patients with Alzheimer's disease (AD). However, the role of NE in AD has not been completely elucidated. Here, Zhang et al. show that β-amyloid oligomers are allosteric ligands of the α2A adrenergic receptor (α2AAR) and modulate NE signaling, redirecting the pathway toward GSK3β activation and subsequent tau hyperphosphorylation. Blocking α2AAR reduced tau phosphorylation and ameliorated pathological and cognitive abnormalities in AD mouse models. The brain noradrenergic system is critical for normal cognition and is affected at early stages in Alzheimer's disease (AD). Here, we reveal a previously unappreciated direct role of norepinephrine signaling in connecting β-amyloid (Aβ) and tau, two key pathological components of AD pathogenesis. Our results show that Aβ oligomers bind to an allosteric site on α2A adrenergic receptor (α2AAR) to redirect norepinephrine-elicited signaling to glycogen synthase kinase 3β (GSK3β) activation and tau hyperphosphorylation. This norepinephrine-dependent mechanism sensitizes pathological GSK3β/tau activation in response to nanomolar accumulations of extracellular Aβ, which is 50- to 100-fold lower than the amount required to activate GSK3β by Aβ alone. The significance of our findings is supported by in vivo evidence in two mouse models, human tissue sample analysis, and longitudinal clinical data. Our study provides translational insights into mechanisms underlying Aβ proteotoxicity, which might have strong implications for the interpretation of Aβ clearance trial results and future drug design and for understanding the selective vulnerability of noradrenergic neurons in AD. [ABSTRACT FROM AUTHOR]

Hsp104 is a yeast member of the Hsp100 family which functions as a molecular chaperone to disaggregate misfolded polypeptides. To understand the mechanism by which the Hsp104 N-terminal domain (NTD) interacts with its peptide substrates, crystal structures of the Hsp104 NTDs from Saccharomyces cerevisiae (ScHsp104NTD) and Candida albicans (CaHsp104NTD) have been determined at high resolution. The structures of ScHsp104NTD and CaHsp104NTD reveal that the yeast Hsp104 NTD may utilize a conserved putative peptide-binding groove to interact with misfolded polypeptides. In the crystal structures ScHsp104NTD forms a homodimer, while CaHsp104NTD exists as a monomer. The consecutive residues Gln105, Gln106 and Lys107, and Lys141 around the putative peptide-binding groove mediate the monomer-monomer interactions within the ScHsp104NTD homodimer. Dimer formation by ScHsp104NTD suggests that the Hsp104 NTD may specifically interact with polyQ regions of prion-prone proteins. The data may reveal the mechanism by which Hsp104 NTD functions to suppress and/or dissolve prions. [ABSTRACT FROM AUTHOR]

PERK is one of the major sensor proteins which can detect the protein-folding imbalance generated by endoplasmic reticulum (ER) stress. It remains unclear how the sensor protein PERK is activated by ER stress. It has been demonstrated that the PERK luminal domain can recognize and selectively interact with misfolded proteins but not native proteins. Moreover, the PERK luminal domain may function as a molecular chaperone to directly bind to and suppress the aggregation of a number of misfolded model proteins. The data strongly support the hypothesis that the PERK luminal domain can interact directly with misfolded proteins to induce ER stress signaling. To illustrate the mechanism by which the PERK luminal domain interacts with misfolded proteins, the crystal structure of the human PERK luminal domain was determined to 3.2 Å resolution. Two dimers of the PERK luminal domain constitute a tetramer in the asymmetric unit. Superimposition of the PERK luminal domain molecules indicated that the β-sandwich domain could adopt multiple conformations. It is hypothesized that the PERK luminal domain may utilize its flexible β-sandwich domain to recognize and interact with a broad range of misfolded proteins. [ABSTRACT FROM AUTHOR]

Mitochondrial preproteins are transported through the translocase of the outer membrane (TOM) complex. Tim50 and Tim23 then transfer preproteins with N-terminal targeting presequences through the intermembrane space (IMS) across the inner membrane. The crystal structure of the IMS domain of Tim50 [Tim50(164-361)] has previously been determined to 1.83 Å resolution. Here, the crystal structure of Tim50(164-361) at 2.67 Å resolution that was crystallized using a different condition is reported. Compared with the previously determined Tim50(164-361) structure, significant conformational changes occur within the protruding β-hairpin of Tim50 and the nearby helix A2. These findings indicate that the IMS domain of Tim50 exhibits significant structural plasticity within the putative presequence-binding groove, which may play important roles in the function of Tim50 as a receptor protein in the TIM complex that interacts with the presequence and multiple other proteins. More interestingly, the crystal packing indicates that helix A1 from the neighboring monomer docks into the putative presequence-binding groove of Tim50(164-361), which may mimic the scenario of Tim50 and the presequence complex. Tim50 may recognize and bind the presequence helix by utilizing the inner side of the protruding β-hairpin through hydrophobic interactions. Therefore, the protruding β-hairpin of Tim50 may play critical roles in receiving the presequence and recruiting Tim23 for subsequent protein translocations. [ABSTRACT FROM AUTHOR]

Mitochondrial precursors are transported through the translocase of the outer membrane (TOM) complex. Tom70/Tom71 is a major surface receptor of the TOM complex for mitochondrial precursors and facilitates Hsp70/Hsp90-escorted precursor translocation into the mitochondrion. Previous structural studies of Tom71 have revealed that it contains an N-terminal and a C-terminal domain and that the two domains may remain in an open conformation when binding to Hsp70/Hsp90. In a newly obtained crystal form of a complex of Tom71 and the Hsp70 C-terminus, the N-terminal domain was found to have rotated about 12° towards the C-terminal domain compared with the previous determined crystal structure of Tom71 in the open conformation. This newly solved structure is defined as the `intermediate conformation'. The domain rearrangements in Tom71 significantly change the surface hydrophobicity and the volume of the precursor-binding pocket. This work suggests that Tom70/Tom71-family members may exhibit structural plasticity from the intermediate conformation to the fully open conformation when complexed with Hsp70/Hsp90. This structural plasticity enables the precursor receptors to accommodate different precursor substrates for mitochondrial translocation. [ABSTRACT FROM AUTHOR]

E. coli Hsp100 ClpB can disaggregate denatured polypeptides by employing ATP hydrolysis. The ClpB N-terminal domain (ClpBN) has been proposed to play important roles in ClpB molecular chaperone activities. We have determined the crystal structure of ClpBN to 1.95 A˚ resolution by MAD methods. The ClpBN monomer contains two subdomains that have similar folds. The crystal structure revealed a hydrophobic groove on the molecular surface. We have constructed ClpB mutants in which the hydrophobic residues within the putative peptide binding groove were replaced by glutamine. These ClpB mutants exhibited severe defects in molecular chaperone activity but retained the wild-type ATPase activity. [Copyright &y& Elsevier]

E. coli Hsp100 ClpB was recently identified as a critical part in a multi-chaperone system to play important roles in protein folding, protein transport and degradation in cell physiology. ClpB contains two nucleotide-binding domains (NBD1 and NBD2) within their primary sequences. NBD1 and NBD2 of ClpB can be classified as members of the large ATPase family known as ATPases associated with various cellular activities (AAA). To investigate how ClpB performs its ATPase activities for its chaperone activity, we have determined the crystal structure of ClpB nucleotide-binding domain 1 (NBD1) by MAD method to 1.80 A˚ resolution. The NBD1 monomer structure contains one domain that comprises 11 α-helices and six β-strands. When compared with the typical AAA structures, the crystal structure of ClpB NBD1 reveals a novel AAA topology with six-stranded β-sheet as its core. The N-terminal portion of NBD1 structure has an extra β-strand flanked by two extra α-helices that are not present in other AAA structures. Moreover, the NBD1 structure does not have a C-terminal helical domain as other AAA proteins do. No nucleotide molecule is bound with ClpB NBD1 in the crystal structure probably due to lack of the C-terminal helix domain in the structure. Isothermal titration calorimetry (ITC) studies of ClpB NBD1 and other ClpB deletion mutations showed that either ClpB NBD1 or NBD2 alone does not bind to nucleotides. However, ClpB NBD2 combined with ClpB C-terminal fragment can interact with one ADP or ATP molecule. ITC data also indicated that full-length ClpB could bind two ADP molecules or one ATP analogue ATPγS molecule. Further ATPase activity studies of ClpB and ClpB deletion mutants showed that only wild-type ClpB have ATPase activity. None of ClpB NBD1 domain, NBD2 domain and NBD2 with C-terminal fragment has detectable ATPase activities. On the basis of our structural and mutagenesis data, we proposed a “see-saw” model to illustrate the mechanisms by which ClpB performs its ATPase activities for chaperone functions. [Copyright &y& Elsevier]

Tail-anchored (TA) proteins represent a unique class of membrane proteins that contain a single C-terminal transmembrane helix. The post-translational insertion of the yeast TA proteins into the ER membrane requires the Golgi ER trafficking (GET) complex which contains Get1, Get2 and Get3. Get3 is an ATPase that recognizes and binds the C-terminal transmembrane domain (TMD) of the TA proteins. We have determined the crystal structures of Get3 from two yeast species, S. cerevisiae and D. hansenii, respectively. These high resolution crystal structures show that Get3 contains a nucleotide-binding domain and a “finger” domain for binding the TA protein TMD. A large hydrophobic groove on the finger domain of S. cerevisiae Get3 structure might represent the binding site for TMD of TA proteins. A hydrophobic helix from a symmetry-related Get3 molecule sits in the TMD-binding groove and mimics the TA binding scenario. Interestingly, the crystal structures of the Get3 dimers from S. cerevisiae and D. hansenii exhibit distinct conformations. The S. cerevisiae Get3 dimer structure does not contain nucleotides and maintains an “open” conformation, while the D. hansenii Get3 dimer structure binds ADP and stays in a “closed” conformation. We propose that the conformational changes to switch the Get3 between the open and closed conformations may facilitate the membrane insertions for TA proteins., Molecular and Cellular Biology

Tail-anchored (TA) proteins contain a single transmembrane domain (TMD) at the C-terminus. The post-translational insertion of TA proteins into the ER membrane requires the cooperation of the Golgi ER-trafficking (GET) complex, which contains Get1, Get2 and Get3. Get3 is a cytosolic ATPase which can recognize and bind the TMD of the TA proteins. Get1 and Get2 are ER transmembrane proteins which can recruit and form a complex with TA-bound Get3. The GET complex carries out an energy-dependent process that facilitates the insertion of the TA-protein TMD into the ER membrane. In order to investigate the mechanism by which the GET complex functions to promote protein insertion into the ER membrane, yeast Get3 has been crystallized. The crystals diffracted to 2.7 Å resolution using a synchrotron X-ray source. The crystals belonged to space group P21212, with unit-cell parameters a = 220.26, b = 112.95, c = 48.27 Å. There is one Get3 dimer in the asymmetric unit, which corresponds to a solvent content of approximately 65%. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), which can promote protein folding and misfolded protein degradation and attenuate protein translation and protein translocation into the ER. P58(IPK) has been proposed to function as a molecular chaperone to maintain protein-folding homeostasis in the ER under normal and stressed conditions. P58(IPK) contains nine TPR motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism by which P58(IPK) functions to promote protein folding within the ER, a P58(IPK) TPR fragment without the C-terminal J-domain was crystallized. The crystals diffract to 2.5 Å resolution using a synchrotron X-ray source. The crystals belong to space group P21, with unit-cell parameters a = 83.53, b = 92.75, c = 84.32 Å, α = 90.00, β = 119.36, γ = 90.00°. There are two P58(IPK) molecules in the asymmetric unit, which corresponds to a solvent content of approximately 60%. Structure determination by MAD methods is under way. [ABSTRACT FROM AUTHOR]

Protein translocations across mitochondrial membranes play critical roles in mitochondrion biogenesis. Protein transport from the cell cytosol to the mitochondrial matrix is carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tim44p is an essential mitochondrial peripheral membrane protein and a major component of the TIM23 translocon. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, the yeast Tim44p was crystallized. The crystals diffract to 3.2 Å using a synchrotron X-ray source and belong to space group P6322, with unit-cell parameters a = 124.25, c = 77.83 Å. There is one Tim44p molecule in one asymmetric unit, which corresponds to a solvent content of approximately 43%. Structure determination by MAD methods is under way. [ABSTRACT FROM AUTHOR]

Heat-shock protein 70 (Hsp70) plays essential roles in a number of cellular processes such as protein folding, assembly and translocation. Heat-shock protein 40 (Hsp40) transiently interacts with Hsp70 and facilitates Hsp70 functions in these processes within cells. Hsp40 recognizes and binds non-native polypeptide and delivers it to Hsp70. Hsp40 can then stimulate the ATPase activity of Hsp70 to refold the polypeptide. To investigate the molecular mechanism by which Hsp40 interacts with Hsp70 to transport the non-native polypeptide, Saccharomyces cerevisiac Hsp40 Sisi C-terminal peptide-binding fragment complexed with Hsp70 Ssa1 C-terminal lid domain has been produced and crystallized. The complex crystals diffract to 3.3 Å and belong to the space group P41212 or P43212, with unit-cell parameters a = 112.17, c = 171.31 Å. Structure determination by the MAD method is under way. [ABSTRACT FROM AUTHOR]

The mechanisms by which Hsp40 functions as a molecular chaperone to recognize and bind nonnative polypeptides is not understood. We have identified a peptide substrate for Ydj1, a member of the type I Hsp40 from yeast. The structure of the Ydj1 peptide binding fragment and its peptide substrate complex was determined to 2.7 A˚ resolution. The complex structure reveals that Ydj1 peptide binding fragment forms an L-shaped molecule constituted by three domains. The domain I exhibits a similar protein folds as domain III while the domain II contains two Zinc finger motifs. The peptide substrate binds Ydj1 by forming an extra β strand with domain I of Ydj1. The Leucine residue in the middle of the peptide substrate GWLYEIS inserts its side chain into a hydrophobic pocket formed on the molecular surface of Ydj1 domain I. The Zinc finger motifs located in the Ydj1 domain II are not in the vicinity of peptide substrate binding site. [Copyright &y& Elsevier]

Abstract: Many mitochondrial proteins are synthesized as preproteins carrying amino-terminal presequences in the cytosol. The preproteins are imported by the translocase of the outer mitochondrial membrane and the presequence translocase of the inner membrane. Tim50 and Tim23 transfer preproteins through the intermembrane space to the inner membrane. We report the crystal structure of the intermembrane space domain of yeast Tim50 to 1.83 Å resolution. A protruding β-hairpin of Tim50 is crucial for interaction with Tim23, providing a molecular basis for the cooperation of Tim50 and Tim23 in preprotein translocation to the protein-conducting channel of the mitochondrial inner membrane. [Copyright &y& Elsevier]

Abstract: P58(IPK) might function as an endoplasmic reticulum molecular chaperone to maintain protein folding homeostasis during unfolded protein responses. P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism by which P58(IPK) functions to promote protein folding within the endoplasmic reticulum, we have determined the crystal structure of P58(IPK) TPR fragment to 2.5 Å resolution by the SAD method. The crystal structure of P58(IPK) revealed three domains (I–III) with similar folds and each domain contains three TPR motifs. An ELISA assay indicated that P58(IPK) acts as a molecular chaperone by interacting with misfolded proteins such as luciferase and rhodanese. The P58(IPK) structure reveals a conserved hydrophobic patch located in domain I that might be involved in binding the misfolded polypeptides. Structure-based mutagenesis for the conserved hydrophobic residues located in domain I significantly reduced the molecular chaperone activity of P58(IPK). [Copyright &y& Elsevier]

Distinct lysine methylation marks on histones create dynamic signatures deciphered by the "effector" modules, although the underlying mechanisms remain unclear. We identified the plant homeodomainand Jumonji C domain-containing protein PHF2 as a novel histone H3K9 demethylase. We show in biochemical and crystallographic analyses that PHF2 recognizes histone H3K4 trimethylation through its plant homeodomain finger and that this interaction is essential for PHF2 occupancy and H3K9 demethylation at rDNA promoters. Our study provides molecular insights into the mechanism by which distinct effector domains within a protein cooperatively modulate the "cross-talk" of histone modifications. [ABSTRACT FROM AUTHOR]

The protein transports from the cell cytosol to the mitochondria matrix are carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tim44p is an essential mitochondrial peripheral membrane protein and a major component of TIM23 translocon. Tim44p can tightly associate with the inner mitochondrial membrane. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, we have determined the crystal structure of the yeast Tim44p C-terminal domain to 3.2Å resolution using the MAD method. The Tim44p C-terminal domain forms a monomer in the crystal structure and contains six α-helices and four antiparallel β-strands. A large hydrophobic pocket was identified on the Tim44p structure surface. The N-terminal helix A1 is positively charged and the helix A1 protrudes out from the Tim44p main body. [Copyright &y& Elsevier]

The molecular chaperone Hsp40 functions as a dimer. The dimer formation is critical for Hsp40 molecular chaperone activity to facilitate Hsp70 to refold non-native polypeptides. We have determined the crystal structure of the C-terminal fragment of yeast Hsp40 Ydj1 that is responsible for Ydj1 dimerization by MAD method. The C-terminal fragment of Ydj1 comprises of the domain III of Ydj1 and the Ydj1 C-terminal dimerization motif. The crystal structure indicates that the dimerization motif of type I Hsp40 Ydj1 differs significantly from that of yeast type II Hsp40. The C terminus of type I Hsp40 Ydj1 from one monomer forms β-strands with the domain III from the other monomer in the homo-dimer. The L372 from Ydj1 C terminus inserts its side-chain into a hydrophobic pocket on domain III. The modeled full-length Ydj1 dimer structure reveals that a large cleft is formed between the two monomers. The domain IIs of Ydj1 monomers that contain the zinc-finger motifs points directly against each other. [Copyright &y& Elsevier]

Golgi homeostasis require the activation of Arf GTPases by the guanine-nucleotide exchange factor requires GBF1, whose recruitment to the Golgi represents a rate limiting step in the process. GBF1 contains a conserved, catalytic, Sec7 domain (Sec7d) and five additional (DCB, HUS, HDS1-3) domains. Herein, we identify the HDS3 domain as essential for GBF1 membrane association in mammalian cells and document the critical role of HDS3 during the development of Drosophila melanogaster . We show that upon binding to Golgi membranes, GBF1 undergoes conformational changes in regions bracketing the catalytic Sec7d. We illuminate GBF1 interdomain arrangements by negative staining electron microscopy of full-length human GBF1 to show that GBF1 forms an anti-parallel dimer held together by the paired central DCB-HUS core, with two sets of HDS1-3 arms extending outward in opposite directions. The catalytic Sec7d protrudes from the central core as a largely independent domain, but is closely opposed to a previously unassigned α-helix from the HDS1 domain. Based on our data, we propose models of GBF1 engagement on the membrane to provide a paradigm for understanding GBF1-mediated Arf activation required for cellular and organismal function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Meissner, Akhmetova, Szul, Viktorova, Sha, Bhatt, Lee, Kahn, Belov, Chesnokov and Sztul.)

Secretory and membrane proteins are folded in the endoplasmic reticulum (ER) prior to their exit. When ER function is disturbed by exogenous and endogenous factors, such as heat shock, ultraviolet radiation, hypoxia, or hypoglycemia, the misfolded proteins may accumulate, promoting ER stress. To rescue this unfavorable situation, the unfolded protein response is activated to reduce misfolded proteins within the ER. Upon ER stress, the ER transmembrane sensor molecules inositol-requiring enzyme 1 (IRE1), RNA-dependent protein kinase (PKR)-like ER kinase (PERK), and activating transcription factor 6, are activated. Here, we discuss the mechanisms of PERK and IRE1 activation and describe two working models for ER stress initiation: the BiP-dependent model and the ligand-driven model. ER stress activation has been linked to multiple diseases, including cancers, Alzheimer's disease, and diabetes. Thus, the regulation of ER stress may provide potential therapeutic targets for these diseases., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

PRKR-like endoplasmic reticulum kinase (PERK) is one of the major sensor proteins that detect protein folding imbalances during endoplasmic reticulum (ER) stress. However, it remains unclear how ER stress activates PERK to initiate a downstream unfolded protein response (UPR). Here, we found that PERK's luminal domain can recognize and selectively interact with misfolded proteins but not with native proteins. Screening a phage-display library, we identified a peptide substrate, P16, of the PERK luminal domain and confirmed that P16 efficiently competes with misfolded proteins for binding this domain. To unravel the mechanism by which the PERK luminal domain interacts with misfolded proteins, we determined the crystal structure of the bovine PERK luminal domain complexed with P16 to 2.8-Å resolution. The structure revealed that PERK's luminal domain binds the peptide through a conserved hydrophobic groove. Substitutions within hydrophobic regions of the PERK luminal domain abolished the binding between PERK and misfolded proteins. We also noted that peptide binding results in major conformational changes in the PERK luminal domain that may favor PERK oligomerization. The structure of the PERK luminal domain-P16 complex suggested stacking of the luminal domain that leads to PERK oligomerization and activation via autophosphorylation after ligand binding. Collectively, our structural and biochemical results strongly support a ligand-driven model in which the PERK luminal domain interacts directly with misfolded proteins to induce PERK oligomerization and activation, resulting in ER stress signaling and the UPR., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Hsp104 is a yeast member of the Hsp100 family which functions as a molecular chaperone to disaggregate misfolded polypeptides. To understand the mechanism by which the Hsp104 N-terminal domain (NTD) interacts with its peptide substrates, crystal structures of the Hsp104 NTDs from Saccharomyces cerevisiae (ScHsp104NTD) and Candida albicans (CaHsp104NTD) have been determined at high resolution. The structures of ScHsp104NTD and CaHsp104NTD reveal that the yeast Hsp104 NTD may utilize a conserved putative peptide-binding groove to interact with misfolded polypeptides. In the crystal structures ScHsp104NTD forms a homodimer, while CaHsp104NTD exists as a monomer. The consecutive residues Gln105, Gln106 and Lys107, and Lys141 around the putative peptide-binding groove mediate the monomer-monomer interactions within the ScHsp104NTD homodimer. Dimer formation by ScHsp104NTD suggests that the Hsp104 NTD may specifically interact with polyQ regions of prion-prone proteins. The data may reveal the mechanism by which Hsp104 NTD functions to suppress and/or dissolve prions.

Sil1 functions as a NEF (nucleotide-exchange factor) for the ER (endoplasmic reticulum) Hsp70 (heat-shock protein of 70 kDa) Bip in eukaryotic cells. Sil1 may catalyse the ADP release from Bip by interacting directly with the ATPase domain of Bip. In the present study we show the complex crystal structure of the yeast Bip and the NEF Sil1 at the resolution of 2.3 Å (1 Å=0.1 nm). In the Sil1-Bip complex structure, the Sil1 molecule acts as a 'clamp' which binds lobe IIb of the Bip ATPase domain. The binding of Sil1 causes the rotation of lobe IIb ~ 13.5° away from the ADP-binding pocket. The complex formation also induces lobe Ib to swing in the opposite direction by ~ 3.7°. These conformational changes open up the nucleotide-binding pocket in the Bip ATPase domain and disrupt the hydrogen bonds between Bip and bound ADP, which may catalyse ADP release. Mutation of the Sil1 residues involved in binding the Bip ATPase domain compromise the binding affinity of Sil1 to Bip, and these Sil1 mutants also abolish the ability to stimulate the ATPase activity of Bip.

The protein translocations across mitochondrial membranes are carried out by specialized complexes, the Translocase of Outer Membrane (TOM) and Translocase of Inner Membrane (TIM). TIM23 translocon is responsible for translocating the mitochondrial matrix proteins across the mitochondrial inner membrane. Tim44 is an essential, peripheral membrane protein in TIM23 complex. Tim44 is tightly associated with the inner mitochondrial membrane on the matrix side. The Tim44 C-Terminal Domain (CTD) functions as an Inner Mitochondrial Membrane (IMM) anchor that recruits the Presequence protein Associated Motor (PAM) to the TIM23 channel. Using X-ray crystallographic and biochemical data, we show that the N-terminal helices A1 and A2 of Tim44 - CTD are crucial for its membrane tethering function. Based on our data, we propose a model showing how the N-terminal A1 and A2 amphipathic helices can either expose their hydrophobic face during membrane binding or conceal it in the soluble form. Therefore, the A1 and A2 helices of Tim44 may function as a membrane sensor.

The endoplasmic reticulum (ER) unfolded protein response (UPR) is comprised of several intracellular signaling pathways that alleviate ER stress. The ER-localized transmembrane kinase PERK is one of three major ER stress transducers. Oligomerization of PERK's N-terminal ER luminal domain by ER stress promotes PERK trans-autophosphorylation of the C-terminal cytoplasmic kinase domain at multiple residues including Thr980 on the kinase activation loop. Activated PERK phosphorylates Ser51 of the α-subunit of translation initiation factor 2 (eIF2α), which inhibits initiation of protein synthesis and reduces the load of unfolded proteins entering the ER. The crystal structure of PERK's kinase domain has been determined to 2.8 Å resolution. The structure resembles the back-to-back dimer observed in the related eIF2α kinase PKR. Phosphorylation of Thr980 stabilizes both the activation loop and helix αG in the C-terminal lobe, preparing the latter for eIF2α binding. The structure suggests conservation in the mode of activation of eIF2α kinases and is consistent with a `line-up' model for PERK activation triggered by oligomerization of its luminal domain.

P58(IPK) has been identified as an ER molecular chaperone to maintain protein-folding homeostasis. P58(IPK) expression can be significantly upregulated during unfolded protein responses (UPR), and it may play important roles in suppressing the ER protein aggregations. To investigate the mechanism how P58(IPK) functions to promote protein folding within ER, we have determined the crystal structure of P58(IPK) TPR domain at 2.5Å resolution. P58(IPK) contains nine TPR motifs and a C-terminal J domain within its primary sequence. The crystal structure of P58(IPK) revealed three subdomains (I, II, and III) with similar folds and each domain contains three TPR motifs. Our data also showed that P58(IPK) acts as a molecular chaperone by interacting with the unfolded proteins such as luciferase, rhodanese, and insulin. The P58(IPK) structure reveals a conserved hydrophobic patch located in subdomain I that may be involved in binding the misfolded polypeptides. We have proposed a working model for P58(IPK) to act together with Bip to prevent protein aggregations and promote protein foldings within ER., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Distinct lysine methylation marks on histones create dynamic signatures deciphered by the "effector" modules, although the underlying mechanisms remain unclear. We identified the plant homeodomain- and Jumonji C domain-containing protein PHF2 as a novel histone H3K9 demethylase. We show in biochemical and crystallographic analyses that PHF2 recognizes histone H3K4 trimethylation through its plant homeodomain finger and that this interaction is essential for PHF2 occupancy and H3K9 demethylation at rDNA promoters. Our study provides molecular insights into the mechanism by which distinct effector domains within a protein cooperatively modulate the "cross-talk" of histone modifications.

The preproteins targeted to the mitochondria are transported through the translocase of the outer membrane complex. Tom70/Tom71 is a major surface receptor of the translocase of the outer membrane complex for mitochondrial preproteins. The preproteins are escorted to Tom70/Tom71 by molecular chaperones Hsp70 and Hsp90. Here we present the high resolution crystal structures of Tom71 and the protein complexes between Tom71 and the Hsp70/Hsp90 C terminus. The crystal structures indicate that Tom70/Tom71 may exhibit two distinct states. In the closed state, the N-terminal domain of Tom70/Tom71 partially blocks the preprotein-binding pocket. In the open state, the N-terminal domain moves away, and the preprotein-binding pocket is fully exposed. The complex formation between the C-terminal EEVD motif of Hsp70/Hsp90 and Tom71 could lock Tom71 in the open state where the preprotein-binding pocket of Tom71 is ready to receive preproteins. The interactions between Hsp70/Hsp90 and Tom71 N-terminal domain generate conformational changes that may increase the volume of the preprotein-binding pocket. The complex formation of Hsp70/Hsp90 and Tom71 also generates significant domain rearrangement within Tom71, which may position the preprotein-binding pocket closer to Hsp70/Hsp90 to facilitate the preprotein transfer from the molecular chaperone to Tom71. Therefore, molecular chaperone Hsp70/Hsp90 may function to prepare the mitochondrial outer membrane receptor Tom71 for preprotein loading.

The mechanism by which Hsp40 and other molecular chaperones recognize and interact with non-native polypeptides is a fundamental question, as is how Hsp40 co-operates with Hsp70 to facilitate protein folding. Years of structural studies of Hsp40 from yeast and other species, conducted using X-ray protein crystallography, NMR and small-angle X-ray scattering, have shed light on the mechanisms how Hsp40 functions as a molecular chaperone and how Hsp40-Hsp70 pair promotes protein folding, protein transport and degradation. This review provides a discussion of recent structural studies of Hsp40s and their functional implications.