Conformational regulation and target-myristoyl switch of calcineurin B homologous protein 3.

Calcineurin B homologous protein 3 (CHP3) is an EF-hand Ca2+-binding protein involved in regulation of cancerogenesis, cardiac hypertrophy, and neuronal development through interactions with sodium/proton exchangers (NHEs) and signalling proteins. While the importance of Ca2+ binding and myristoylation for CHP3 function has been recognized, the underlying molecular mechanism remained elusive. In this study, we demonstrate that Ca2+ binding and myristoylation independently affect the conformation and functions of human CHP3. Ca2+ binding increased local flexibility and hydrophobicity of CHP3 indicative of an open conformation. The Ca2+-bound CHP3 exhibited a higher affinity for NHE1 and associated stronger with lipid membranes compared to the Mg2+-bound CHP3, which adopted a closed conformation. Myristoylation enhanced the local flexibility of CHP3 and decreased its affinity to NHE1 independently of the bound ion, but did not affect its binding to lipid membranes. The data exclude the proposed Ca2+-myristoyl switch for CHP3. Instead, a Ca2+-independent exposure of the myristoyl moiety is induced by binding of the target peptide to CHP3 enhancing its association to lipid membranes. We name this novel regulatory mechanism 'target-myristoyl switch'. Collectively, the interplay of Ca2+ binding, myristoylation, and target binding allows for a context-specific regulation of CHP3 functions.

Stabilization of membrane topologies by proteinaceous remorin scaffolds.

In plants, the topological organization of membranes has mainly been attributed to the cell wall and the cytoskeleton. Additionally, few proteins, such as plant-specific remorins have been shown to function as protein and lipid organizers. Root nodule symbiosis requires continuous membrane re-arrangements, with bacteria being finally released from infection threads into membrane-confined symbiosomes. We found that mutations in the symbiosis-specific SYMREM1 gene result in highly disorganized perimicrobial membranes. AlphaFold modelling and biochemical analyses reveal that SYMREM1 oligomerizes into antiparallel dimers and may form a higher-order membrane scaffolding structure. This was experimentally confirmed when expressing this and other remorins in wall-less protoplasts is sufficient where they significantly alter and stabilize de novo membrane topologies ranging from membrane blebs to long membrane tubes with a central actin filament. Reciprocally, mechanically induced membrane indentations were equally stabilized by SYMREM1. Taken together we describe a plant-specific mechanism that allows the stabilization of large-scale membrane conformations independent of the cell wall.

Calcium affects CHP1 and CHP2 conformation and their interaction with sodium/proton exchanger 1.

Calcineurin B homologous proteins (CHPs) belong to the EF-hand Ca2+ -binding protein (EFCaBP) family. They have multiple important functions including the regulation of the Na+ /H+ exchanger 1 (NHE1). The human isoforms CHP1 and CHP2 share high sequence similarity, but have distinct expression profiles with CHP2 levels for instance increased in malignant cells. These CHPs bind Ca2+ with high affinity. Biochemical data indicated that Ca2+ can regulate their functions. Experimental evidence for Ca2+ -modulated structural changes was lacking. With a newly established fluorescent probe hydrophobicity (FPH) assay, we detected Ca2+ -induced conformational changes in both CHPs. These changes are in line with an opening of their hydrophobic pocket that binds the CHP-binding region (CBD) of NHE1. Whereas the pocket is closed in the absence of Ca2+ in CHP2, it is still accessible for the dye in CHP1. Both CHPs interacted with CBD in the presence and absence of Ca2+ . Isothermal titration calorimetry (ITC) analysis revealed high binding affinity for both CHPs to CBD with equilibrium dissociation constants (KD s) in the nanomolar range. The KD for CHP1:CBD was not affected by Ca2+ , whereas Ca2+ -depletion increased the KD 7-fold for CHP2:CBD showing a decreased affinity. The data indicate an isoform specific regulatory interaction of CHP1 and CHP2 with NHE1.

Regulation of small heat-shock proteins by hetero-oligomer formation.

Small heat-shock proteins (sHsps) compose the most widespread family of molecular chaperones. The human genome encodes 10 different sHsps (HspB1-10). It has been shown that HspB1 (Hsp27), HspB5 (αB-crystallin), and HspB6 (Hsp20) can form hetero-oligomers in vivo However, the impact of hetero-oligomerization on their structure and chaperone mechanism remains enigmatic. Here, we analyzed hetero-oligomer formation in human cells and in vitro using purified proteins. Our results show that the effect of hetero-oligomer formation on the composition of the sHsp ensembles and their chaperone activities depends strongly on the respective sHsps involved. We observed that hetero-oligomer formation between HspB1 and HspB5 leads to an ensemble that is dominated by species larger than the individual homo-oligomers. In contrast, the interaction of dimeric HspB6 with either HspB1 or HspB5 oligomers shifted the ensemble toward smaller oligomers. We noted that the larger HspB1-HspB5 hetero-oligomers are less active and that HspB6 activates HspB5 by dissociation to smaller oligomer complexes. The chaperone activity of HspB1-HspB6 hetero-oligomers, however, was modulated in a substrate-specific manner, presumably due to the specific enrichment of an HspB1-HspB6 heterodimer. These heterodimeric species may allow the tuning of the chaperone properties toward specific substrates. We conclude that sHsp hetero-oligomerization exerts distinct regulatory effects depending on the sHsps involved.

The structure and oxidation of the eye lens chaperone αA-crystallin.

The small heat shock protein αA-crystallin is a molecular chaperone important for the optical properties of the vertebrate eye lens. It forms heterogeneous oligomeric ensembles. We determined the structures of human αA-crystallin oligomers by combining cryo-electron microscopy, cross-linking/mass spectrometry, NMR spectroscopy and molecular modeling. The different oligomers can be interconverted by the addition or subtraction of tetramers, leading to mainly 12-, 16- and 20-meric assemblies in which interactions between N-terminal regions are important. Cross-dimer domain-swapping of the C-terminal region is a determinant of αA-crystallin heterogeneity. Human αA-crystallin contains two cysteines, which can form an intramolecular disulfide in vivo. Oxidation in vitro requires conformational changes and oligomer dissociation. The oxidized oligomers, which are larger than reduced αA-crystallin and destabilized against unfolding, are active chaperones and can transfer the disulfide to destabilized substrate proteins. The insight into the structure and function of αA-crystallin provides a basis for understanding its role in the eye lens.

Calcineurin B homologous protein 3 binds with high affinity to the CHP binding domain of the human sodium/proton exchanger NHE1.

The Na+/H+ exchanger NHE1 is critical for cell vitality as it controls intracellular pH and cell volume. Its functionality is influenced by calcineurin B homologous proteins (CHPs). The human isoform CHP3 is important for transport of NHE1 to the plasma membrane and for its activity. Here, we characterized the binding interaction of human CHP3 with the regulatory domain of NHE1. The exact binding site of CHP3 was previously debated. CHP3 as well as both regions of NHE1 in question were produced and purified. CHP3 specifically formed stable complexes with the CHP-binding region (CBD) of NHE1 (residues 503-545) in size-exclusion chromatography (SEC), but not with the C-terminal region (CTD, residues 633-815). CTD was functional as shown by Ca2+-dependent binding of calmodulin in SEC analysis. CHP3 bound with high affinity to CBD with an equilibrium dissociation constant (KD) of 56 nM determined by microscale thermophoresis. The high affinity was substantiated by isothermal calorimetry analysis (KD = 3 nM), which also revealed that the interaction with CBD is strongly exothermic (ΔG° = -48.6 kJ/mol, ΔH = -75.3 kJ/mol, -TΔS° = 26.7 kJ/mol). The data provide insights in the molecular mechanisms that underlie the regulatory interaction of CHP3 and NHE1 and more general of calcineurin homologous proteins with their target proteins.

The Chaperone Activity and Substrate Spectrum of Human Small Heat Shock Proteins.

Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones that suppress the unspecific aggregation of miscellaneous proteins. Multicellular organisms contain a large number of different sHsps, raising questions as to whether they function redundantly or are specialized in terms of substrates and mechanism. To gain insight into this issue, we undertook a comparative analysis of the eight major human sHsps on the aggregation of both model proteins and cytosolic lysates under standardized conditions. We discovered that sHsps, which form large oligomers (HspB1/Hsp27, HspB3, HspB4/αA-crystallin, and HspB5/αB-crystallin) are promiscuous chaperones, whereas the chaperone activity of the other sHsps is more substrate-dependent. However, all human sHsps analyzed except HspB7 suppressed the aggregation of cytosolic proteins of HEK293 cells. We identified ∼1100 heat-sensitive HEK293 proteins, 12% of which could be isolated in complexes with sHsps. Analysis of their biochemical properties revealed that most of the sHsp substrates have a molecular mass from 50 to 100 kDa and a slightly acidic pI (5.4-6.8). The potency of the sHsps to suppress aggregation of model substrates is correlated with their ability to form stable substrate complexes; especially HspB1 and HspB5, but also B3, bind tightly to a variety of proteins, whereas fewer substrates were detected in complex with the other sHsps, although these were also efficient in preventing the aggregation of cytosolic proteins.

Medical implications of understanding the functions of human small heat shock proteins.

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that are implicated in a variety of diseases. Upon stress, they stabilize unfolding proteins and prevent them from aggregating. However, under physiological conditions without severe stress, some sHsps interact with other proteins. In a perspective view, their ability to bind specific client proteins might allow them to fine-tune the availability of the client for other, client-dependent cellular processes. Additionally, some sHsps seem to interact with specific co-chaperones. These co-chaperones are usually part of large protein machineries that are functionally modulated upon sHsps interaction. Finally, secreted human sHsps seem to interact with receptor proteins, potentially as signal molecules transmitting the stress status from one cell to another. This review focuses on the mechanistic description of these different binding modes for human sHsps and how this might help to understand and modulate the function of sHsps in the context of disease.

Utilization of fluorescent chimeras for investigation of heterooligomeric complexes formed by human small heat shock proteins.

Fluorescent chimeras composed of enhanced cyan (or enhanced yellow) fluorescent proteins (ECFP or EYFP) and one of the four human small heat shock proteins (HspB1, HspB5, HspB6 or HspB8) were expressed in E. coli and purified. Fluorescent chimeras were used for investigation of heterooligomeric complexes formed by different small heat shock proteins (sHsp) and for analysis of their subunit exchange. EYFP-HspB1 and ECFP-HspB6 form heterooligomeric complex with apparent molecular weight of ∼280 kDa containing equimolar quantities of both sHsp. EYFP-HspB5 and ECFP-HspB6 formed heterogeneous oligomeric complexes. Fluorescent proteins inside heterooligomeric complexes formed by HspB1/HspB6 and HspB5/HspB6 chimeras are closely located, making possible effective fluorescence resonance energy transfer (FRET). Neither the wild type HspB8 nor its fluorescent chimeras were able to form stable heterooligomeric complexes with the wild type HspB1 and HspB5. Homo- and hetero-FRET was used for analysis of subunit exchange of small heat shock proteins. The apparent rate constant of subunit exchange was temperature-dependent and was higher for HspB6 forming small oligomers than for HspB1 forming large oligomers. Replacement induced by homologous subunits was more rapid than the replacement induced by heterologous subunits of small heat shock proteins. Fusion of fluorescent proteins might affect oligomeric structure of small heat shock proteins, however fluorescent chimeras can be useful for investigation of heterooligomeric complexes formed by sHsp and for analysis of kinetics of their subunit exchange.

Heterooligomeric complexes of human small heat shock proteins.

Oligomeric association of human small heat shock proteins HspB1, HspB5, HspB6 and HspB8 was analyzed by means of size-exclusion chromatography, analytical ultracentrifugation and chemical cross-linking. Wild-type HspB1 and Cys mutants of HspB5, HspB6 and HspB8 containing a single Cys residue in position homologous to that of Cys137 of human HspB1 were able to generate heterodimers cross-linked by disulfide bond. Cross-linked heterodimers between HspB1/HspB5, HspB1/HspB6 and HspB5/HspB6 were easily produced upon mixing, whereas formation of any heterodimers with participation of HspB8 was significantly less efficient. The size of heterooligomers formed by HspB1/HspB6 and HspB5/HspB6 was different from the size of the corresponding homooligomers. Disulfide cross-linked homodimers of small heat shock proteins were unable to participate in heterooligomer formation. Thus, monomers can be involved in subunit exchange leading to heterooligomer formation and restriction of flexibility induced by disulfide cross-linking prevents subunit exchange.

The role of intrinsically disordered regions in the structure and functioning of small heat shock proteins.

Small heat shock proteins (sHsp) form a large ubiquitous family of proteins expressed in all phyla of living organisms. The members of this family have low molecular masses (13-43 kDa) and contain a conservative α-crystallin domain. This domain (about 90 residues) consists of several ß-strands forming two ß-sheets packed in immunoglobulinlike manner. The α-crystallin domain plays an important role in formation of stable sHsp dimers, which are the building blocks of the large sHsp oligomers. A large N-terminal domain and a short C-terminal extension flank the α-crystallin domain. Both the N-terminal domain and the C-terminal extension are flexible, susceptible to proteolysis, prone to posttranslational modifications, and are predominantly intrinsically disordered. Differently oriented N-terminal domains interact with each other, with the core α-crystallin domain of the same or neighboring dimers and play important role in formation of large sHsp oligomers. Phosphorylation of certain sites in the N-terminal domain affects the sHsp quaternary structure, the sHsp interaction with target proteins and the sHsp chaperone-like activity. The C-terminal extension often carrying the conservative tripeptide (I/V/L)-X-(I/V/L) is capable of binding to a hydrophobic groove on the surface of the core α-crystallin domain of neighboring dimer, thus affecting the plasticity and the overall structure of sHsp oligomers. The Cterminal extension interacts with target proteins and affects their interaction with the α-crystallin domain increasing solubility of the complexes formed by sHsp and their targets. Thus, disordered N- and C-terminal sequences play important role in the structure, regulation and functioning of sHsp.

Expression, purification and some properties of fluorescent chimeras of human small heat shock proteins.

Small heat shock proteins (sHsp) are ubiquitously expressed in all human tissues and have an important housekeeping role in preventing the accumulation of aggregates of improperly folded or denatured proteins. They also participate in the regulation of the cytoskeleton, proliferation, apoptosis and many other vital processes. Fluorescent chimeras composed of sHsp and enhanced fluorescent proteins have been used to determine the intracellular locations of small heat shock proteins and to analyse the hetero-oligomeric complexes formed by different sHsp. However, the biochemical properties and chaperone-like activities of these chimeras have not been investigated. To determine the properties of these chimeras, we fused enhanced yellow and cyan fluorescent proteins (EYFP and ECFP) to the N-termini of four ubiquitously expressed human small heat shock proteins: HspB1, HspB5, HspB6, and HspB8. The eight fluorescent chimeras of small heat shock proteins and isolated fluorescent proteins were expressed in Escherichia coli. The chimeric proteins were isolated and purified via ammonium sulphate fractionation, ion exchange and size-exclusion chromatography. This method provided 20-100 mg of fluorescent chimeras from 1L of bacterial culture. The spectral properties of the chimeras were similar to those of the isolated fluorescent proteins. The fusion of fluorescent proteins to HspB6 and HspB8, which typically form dimers, did not affect their quaternary structures. Oligomers of the fluorescent chimeras of HspB1 and HspB5 were less stable and contained fewer subunits than oligomers formed by the wild-type proteins. Fusion with EYFP decreased the chaperone-like activity of HspB5 and HspB6 whereas fusion with ECFP increased chaperone-like activity. All fluorescent chimeras of HspB1 and HspB8 had higher chaperone-like activity than the wild-type proteins. Thus, although fluorescent chimeras are useful for many purposes, the fluorescent proteins used to form these chimeras may affect certain important properties of sHsp.

Large potentials of small heat shock proteins.

Modern classification of the family of human small heat shock proteins (the so-called HSPB) is presented, and the structure and properties of three members of this family are analyzed in detail. Ubiquitously expressed HSPB1 (HSP27) is involved in the control of protein folding and, when mutated, plays a significant role in the development of certain neurodegenerative disorders. HSPB1 directly or indirectly participates in the regulation of apoptosis, protects the cell against oxidative stress, and is involved in the regulation of the cytoskeleton. HSPB6 (HSP20) also possesses chaperone-like activity, is involved in regulation of smooth muscle contraction, has pronounced cardioprotective activity, and seems to participate in insulin-dependent regulation of muscle metabolism. HSPB8 (HSP22) prevents accumulation of aggregated proteins in the cell and participates in the regulation of proteolysis of unfolded proteins. HSPB8 also seems to be directly or indirectly involved in regulation of apoptosis and carcinogenesis, contributes to cardiac cell hypertrophy and survival and, when mutated, might be involved in development of neurodegenerative diseases. All small heat shock proteins play important "housekeeping" roles and regulate many vital processes; therefore, they are considered as attractive therapeutic targets.

The pivotal role of the beta 7 strand in the intersubunit contacts of different human small heat shock proteins.

Human alpha B-crystallin and small heat shock proteins HspB6 and HspB8 were mutated so that all endogenous Cys residues were replaced by Ser and the single Cys residue was inserted in a position homologous to that of Cys137 of human HspB1, i.e. in a position presumably located in the central part of beta 7 strand of the alpha-crystallin domain. The secondary, tertiary, and quaternary structures of thus obtained Cys-mutants as well as their chaperone-like activity were similar to those of their wild-type counterparts. Mild oxidation of Cys-mutants leads to formation of disulfide bond crosslinking neighboring monomers thus indicating participation of the beta 7 strand in intersubunit interaction. Oxidation weakly affects the secondary and tertiary structure, does not affect the quaternary structure of alpha B-crystallin and HspB6, and shifts equilibrium between monomer and dimer of HspB8 towards dimer formation. It is concluded that the beta 7 strand participates in the intersubunit interaction of four human small heat shock proteins (alpha B-crystallin, HspB1, HspB6, HspB8) having different structure of beta2 strand of alpha-crystallin domain and different length and composition of variable N- and C-terminal tails.