The Hsc70 system maintains the synaptic SNARE protein SNAP-25 in an assembly-competent state and delays its aggregation.

The complex mechanism of synaptic vesicle fusion with the plasma membrane for neurotransmitter release is initiated by the formation of the SNARE complex at the presynaptic terminal of the neuron. The SNARE complex is composed of four helices contributed by three proteins: one from syntaxin (localized at the plasma membrane), one from synaptobrevin (localized at the synaptic vesicle), and two from the intrinsically disordered and aggregation-prone synaptosomal-associated 25 kDa protein (SNAP-25), which is localized to the plasma membrane by virtue of palmitoylation of cysteine residues. The fusion process is tightly regulated and requires the constitutively expressed Hsp70 chaperone (Hsc70) and its J-protein co-chaperone CSPα.

Insights into the interaction between UGGT, the gatekeeper of folding in the ER, and its partner, the selenoprotein SEP15.

The enzyme UDP-glucose: glycoprotein glucosyltransferase (UGGT) is the gatekeeper of protein folding within the endoplasmic reticulum (ER). One-third of the human proteome traverses the ER where folding and maturation are facilitated by a complex protein homeostasis network. Both glycan modifications and disulfide bonds are of key importance in the maturation of these ER proteins.

New insights into the structure and function of the complex between the Escherichia coli Hsp70, DnaK, and its nucleotide-exchange factor, GrpE.

The 70 kDa heat shock proteins (Hsp70s) play a pivotal role in many cellular functions using allosteric communication between their nucleotide-binding domain (NBD) and substrate-binding domain, mediated by an interdomain linker, to modulate their affinity for protein clients. Critical to modulation of the Hsp70 allosteric cycle, nucleotide-exchange factors (NEFs) act by a conserved mechanism involving binding to the ADP-bound NBD and opening of the nucleotide-binding cleft to accelerate the release of ADP and binding of ATP. The crystal structure of the complex between the NBD of the Escherichia coli Hsp70, DnaK, and its NEF, GrpE, was reported previously, but the GrpE in the complex carried a point mutation (G122D).

Computationally-Aided Modeling of Hsp70-Client Interactions: Past, Present, and Future.

Hsp70 molecular chaperones play central roles in maintaining a healthy cellular proteome. Hsp70s function by binding to short peptide sequences in incompletely folded client proteins, thus preventing them from misfolding and/or aggregating, and in many cases holding them in a state that is competent for subsequent processes like translocation across membranes. There is considerable interest in predicting the sites where Hsp70s may bind their clients, as the ability to do so sheds light on the cellular functions of the chaperone.

There are more Hsp90 chaperone mechanisms in heaven and earth, dear reader, than are dreamt of in your philosophy.

Dahiya et al. (2022) and Biebl et al. (2022) present mechanistic insights into the Hsp40/Hsp70/Hsp90 chaperone teamwork and the co-chaperones that participate in this network.

Physics-based modeling provides predictive understanding of selectively promiscuous substrate binding by Hsp70 chaperones.

To help cells cope with protein misfolding and aggregation, Hsp70 molecular chaperones selectively bind a variety of sequences ("selective promiscuity"). Statistical analyses from substrate-derived peptide arrays reveal that DnaK, the E. coli Hsp70, binds to sequences containing three to five branched hydrophobic residues, although otherwise the specific amino acids can vary considerably.

Selective promiscuity in the binding of Hsp70 to an unfolded protein.

Heat shock protein 70 (Hsp70) chaperones bind many different sequences and discriminate between incompletely folded and folded clients. Most research into the origins of this "selective promiscuity" has relied on short peptides as substrates to dissect the binding, but much less is known about how Hsp70s bind full-length client proteins. Here, we connect detailed structural analyses of complexes between the Hsp70 (DnaK) substrate-binding domain (SBD) and peptides encompassing five potential binding sites in the precursor to alkaline phosphatase (proPhoA) with SBD binding to full-length unfolded proPhoA.

Hsp70 molecular chaperones: multifunctional allosteric holding and unfolding machines.

The Hsp70 family of chaperones works with its co-chaperones, the nucleotide exchange factors and J-domain proteins, to facilitate a multitude of cellular functions. Central players in protein homeostasis, these jacks-of-many-trades are utilized in a variety of ways because of their ability to bind with selective promiscuity to regions of their client proteins that are exposed when the client is unfolded, either fully or partially, or visits a conformational state that exposes the binding region in a regulated manner. The key to Hsp70 functions is that their substrate binding is transient and allosterically cycles in a nucleotide-dependent fashion between high- and low-affinity states.

Allosteric landscapes of eukaryotic cytoplasmic Hsp70s are shaped by evolutionary tuning of key interfaces.

The 70-kDa heat shock proteins (Hsp70s) are molecular chaperones that perform a wide range of critical cellular functions. They assist in the folding of newly synthesized proteins, facilitate assembly of specific protein complexes, shepherd proteins across membranes, and prevent protein misfolding and aggregation. Hsp70s perform these functions by a conserved mechanism that relies on allosteric cycles of nucleotide-modulated binding and release of client proteins.

N-glycosylation Triggers a Dual Selection Pressure in Eukaryotic Secretory Proteins.

Nearly one third of the eukaryotic proteome traverses the secretory pathway and most of these proteins are N-glycosylated in the lumen of the endoplasmic reticulum. N-glycans fulfill multiple structural and biological functions, and are crucial for productive folding of many glycoproteins. N-glycosylation involves the attachment of an oligosaccharide to selected asparagine residues in the sequence N-X-S/T (X ≠ P), a motif known as an N-glycosylation'sequon'.

Key features of an Hsp70 chaperone allosteric landscape revealed by ion-mobility native mass spectrometry and double electron-electron resonance.

Proteins are dynamic entities that populate conformational ensembles, and most functions of proteins depend on their dynamic character. Allostery, in particular, relies on ligand-modulated shifts in these conformational ensembles. Hsp70s are allosteric molecular chaperones with conformational landscapes that involve large rearrangements of their two domains ( the nucleotide-binding domain and substrate-binding domain) in response to adenine nucleotides and substrates.

Division of Labor: ER-Resident BiP Co-Chaperones Match Substrates to Fates Based on Specific Binding Sequences.

In this issue of Molecular Cell, Behnke et al. (2016) describe a novel cell-based peptide-binding assay and use it to analyze the binding specificities of the endoplasmic reticulum Hsp70 chaperone and its co-chaperones and to probe their different roles in protein quality control.

Hsp70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to Hsp90.

Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR).

How hsp70 molecular machines interact with their substrates to mediate diverse physiological functions.

Hsp70 molecular chaperones are implicated in a wide variety of cellular processes, including protein biogenesis, protection of the proteome from stress, recovery of proteins from aggregates, facilitation of protein translocation across membranes, and more specialized roles such as disassembly of particular protein complexes. It is a fascinating question to ask how the mechanism of these deceptively simple molecular machines is matched to their roles in these wide-ranging processes. The key is a combination of the nature of the recognition and binding of Hsp70 substrates and the impact of Hsp70 action on their substrates.

An interdomain energetic tug-of-war creates the allosterically active state in Hsp70 molecular chaperones.

The allosteric mechanism of Hsp70 molecular chaperones enables ATP binding to the N-terminal nucleotide-binding domain (NBD) to alter substrate affinity to the C-terminal substrate-binding domain (SBD) and substrate binding to enhance ATP hydrolysis. Cycling between ATP-bound and ADP/substrate-bound states requires Hsp70s to visit a state with high ATPase activity and fast on/off kinetics of substrate binding. We have trapped this "allosterically active" state for the E.

Efficient isolation of Pseudomonas aeruginosa type III secretion translocators and assembly of heteromeric transmembrane pores in model membranes.

Translocation of bacterial toxins or effectors into host cells using the type III secretion (T3S) system is a conserved mechanism shared by many Gram-negative pathogens. Pseudomonas aeruginosa injects different proteins across the plasma membrane of target cells, altering the normal metabolism of the host. Protein translocation presumably occurs through a proteinaceous transmembrane pore formed by two T3S secreted protein translocators, PopB and PopD.

Segmental isotopic labeling of the Hsp70 molecular chaperone DnaK using expressed protein ligation.

Introducing biophysical labels into specific regions of large and dynamic multidomain proteins greatly facilitates mechanistic analysis. Ligation of expressed domains that are labeled in a desired manner before assembly of the intact molecular machine provides such a strategy. We have elaborated an experimental route using expressed protein ligation (EPL) to create an Hsp70 molecular chaperone (the E.

Exploring the interactions between signal sequences and E. coli SRP by two distinct and complementary crosslinking methods.

Photoaffinity crosslinking comprises a group of invaluable techniques used to investigate in detail a binding interaction between two polypeptides. As the diverse photo crosslinking techniques available display inherent differences, the method of choice will provide specific information about a particular system under study. We used two complementary crosslinking approaches: photo-induced crosslinking of unmodified proteins (PICUP) and benzophenone-mediated (BPM) crosslinking to extensively examine the interaction between the signal recognition particle (SRP) and signal sequences.

Stability and lability of circadian period of gene expression in the cyanobacterium Synechococcus elongatus.

Molecular aspects of the circadian clock in the cyanobacterium Synechococcus elongatus have been described in great detail. Three-dimensional structures have been determined for the three proteins, KaiA, KaiB and KaiC, that constitute a central oscillator of the clock. Moreover, a temperature-compensated circadian rhythm of KaiC phosphorylation can be reconstituted in vitro with the addition of KaiA, KaiB and ATP.

Use of synthetic signal sequences to explore the protein export machinery.

The information for correct localization of newly synthesized proteins in both prokaryotes and eukaryotes resides in self-contained, often transportable targeting sequences. Of these, signal sequences specify that a protein should be secreted from a cell or incorporated into the cytoplasmic membrane. A central puzzle is presented by the lack of primary structural homology among signal sequences, although they share common features in their sequences.

Specialized techniques for site-directed mutagenesis in cyanobacteria.

Synechococcus elongatus PCC 7942 is an excellent model system for studying the molecular mechanism of the circadian clock in cyanobacteria. The "plastic" genetic characteristics of this organism have facilitated the development of various methods for mutagenesis of its chromosome. These methods are based on homologous recombination between the chromosome and foreign DNA, introduced to the cyanobacteria by either transformation or conjugation.

Detection of rhythmic bioluminescence from luciferase reporters in cyanobacteria.

The unicellular cyanobacterium Synechococcus elongatus PCC 7942 is the model organism for studying prokaryotic circadian rhythms. Although S. elongatus does not display an easily measurable overt circadian behavior, its gene expression is under circadian control; hence, a "behavior" is created by linking a cyanobacterial promoter to either the bacterial luxAB or firefly luc luciferase genes to create reporter fusions whose activity can be easily monitored by bioluminescence.