Specific Sequences in the N-terminal Domain of Human Small Heat Shock Protein HSPB6 Dictate Preferential Heterooligomerization with the Orthologue HSPB1

Small heat-shock proteins (sHSPs) are a conserved group of molecular chaperones with important roles in cellular proteostasis. Although sHSPs are characterized by their small monomeric weight, they typically assemble into large polydisperse oligomers that vary in both size and shape but are principally composed of dimeric building blocks. These assemblies can include different sHSP orthologues, creating additional complexity that may affect chaperone activity. However, the structural and functional properties of such hetero-oligomers are poorly understood. We became interested in hetero-oligomer formation between human heat-shock protein family B (small) member 1 (HSPB1) and HSPB6, which are both highly expressed in skeletal muscle. When mixed in vitro, these two sHSPs form a polydisperse oligomer array composed solely of heterodimers, suggesting preferential association that is determined at the monomer level. Previously, we have shown that the sHSP N-terminal domains (NTDs), which have a high degree of intrinsic disorder, are essential for the biased formation. Here we employed iterative deletion mapping to elucidate how the NTD of HSPB6 influences its preferential association with HSPB1 and show that this region has multiple roles in this process. First, the highly conserved motif RLFDQXFG is necessary for subunit exchange among oligomers. Second, a site ∼20 residues downstream of this motif determines the size of the resultant hetero-oligomers. Third, a region unique to HSPB6 dictates the preferential formation of heterodimers. In conclusion, the disordered NTD of HSPB6 helps regulate the size and stability of hetero-oligomeric complexes, indicating that terminal sHSP regions define the assembly properties of these proteins

Biochemical and biophysical characterization of human small heat shock proteins

Small heat shock proteins play an important role in maintaining the quality of all proteins in a cell, both under normal and stress conditions and are part of a large network called the protein quality control network. sHSPs can be considered as a first line of defense against unfolding protein species under stress conditions, this is because of their ATP-independent mode of action. These chaperones can bind partially unfolded proteins and trap them in a folding competent state. Because of their importance in proteostasis, it is not surprising that these proteins have been associated with many human diseases. Many sHSPs co-localize with amyloid deposits in debilitating neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease. Mutation of sHSPs lead to congenital forms of myopathies, neuropathies and cataract and upregulation of sHSP genes is seen during cancer development. This makes them an interesting target for developing new drugs, although to date attempts have been limited. All sHSPs share the same architectural arrangement: a central conserved domain of approximately 90 residues called the α-crystallin domain, flanked by unstructured and N- and C-terminal arms that are considered to be poorly conserved. All sHSPs assemble into dimers via their α-crystallin domain and assemble further into large oligomers that can comprise up to 40 subunits. This assembly is dependent on interactions mediated by the N- and C-terminal arms, although details about the interacting residues are missing. Most mammalian sHSPs assemble into polydisperse oligomers that can vary in both size, shape and the number of subunits and can even contain different sHSPs expressed in the same tissue, which makes these proteins nearly impossible to crystallize. The chaperone activity of these proteins is also linked to the N-terminal region, but due to the low conservation and lack of secondary structure, studies regarding the structure and function of these proteins have been an arduous task. This is mainly due to the overlapping role the N-terminal region plays in both structure and function, so truncation or mutation usually has a concerted effect on size and chaperone activity, making it hard to delineate the sequence properties. This research project focuses on characterizing the structure and function two human small heat shock proteins, HSPB1 and HSPB6. Both proteins reach high levels of expression in smooth muscle tissue and are known to hetero-oligomerize. In order to gain insights into the specific sequences necessary for both chaperoning and assembly, we have used HSPB6 as a model sHSP. This particular sHSP does not form the typical large oligomeric assemblies but only forms dimers in solution. This property makes HSPB6 an excellent model to study the functional epitopes, as mutation or truncation is unlikely to affect the size of the protein. Furthermore, this protein hetero-oligomerizes with HSPB1, so the sequences involved in this interaction can also be investigated. As a first objective, the sequence determinants that define the chaperone activity of HSPB6 were determined by creating a library of deletion constructs in which 10 amino acids were deleted stepwise. These truncations were all characterized using size-exclusion chromatography and small-angle X-ray scattering to determine the effect of truncation on the structure and we have found that all were still dimeric in solution. Therefore, the activity of each was assessed using standard chaperone assays in which an aggregation-prone protein was incubated with different amounts of a sHSP. All of the truncations, except for a complete removal of the NTR were still capable of protecting insulin and yeast alcohol dehydrogenase – two standard substrates – from aggregation. Deletion of residues 41 to 60 of the NTR led to a reduced activity when compared to the wild type, although differences in the chaperoning profile for the two substrates were observed. This suggests that likely multiple regions within the NTR are necessary for chaperoning and that these regions may even be redundant, or display substrate specificity. Surprisingly, deletion of a central conserved stretch in the NTR, encompassing residues 31-40 led to a vast increase in activity. Further mapping using smaller deletions and point mutations, have shown that the glutamate at position 31 functions as a negative regulator of activity, and that the residues surrounding it also affect its regulatory function. Even though HSPB6 does not assemble into multisubunit oligomers, it does display concentration dependent self-association in solution. This non-ideal behavior is also regulated by the conserved residues found in the 31 to 35 region, where the phenylalanine at position 33 regulates this specific property. Again, this feature is also modulated by the surrounding residues, as mutation of Glu31 leads to increased self-association. It thus seems that residues in the N-terminal region of HSPB6 that are conserved throughout metazoan sHSPs, have an important regulatory role. The results described in Chapter 3, hint at a fine-tuned interplay between these residues in regulating both assembly and function of sHSPs. To further investigate the effect of the NTR in defining the structure of sHSPs, the same library of deletion constructs and mutants was used to assess hetero-oligomerization with HSPB1. HSPB6 and HSPB1 have been shown to form hetero-oligomers in vivo and in vitro. Size exclusion chromatography and disulfide crosslinking (using a mutant of HSPB6 that is capable of forming a disulfide crosslinked dimer) have shown that this complex consists of a highly polydisperse mix containing two main species of approximately 500 kDa and 150 kDa, both built up of heterodimers. We have shown, using native mass spectrometry that this heterocomplex is built up of 100% heterodimers, even though both proteins on their own are capable of exchanging subunits in a stochastic fashion. Large truncations, where only the NTR (ΔN) or both the NTR and CTR (ACD) was removed, showed that the NTR is the key player in dictating this preferential interaction between the wild type proteins, as both truncations led to a mere stochastic exchange between the two sHSPs. The conserved region, identified as negative regulator of activity in HSPB6 before, was also found to be essential for complex formation with HSPB1. Deletion of this region in HSPB6 prevented the formation of a complex, whereas deletion of the residues adjacent to this region (36 to 40) were necessary for the preferential interaction between HSPB1 and HSPB6. These results are described in Chapter 4. Thus, overall we have found an essential role for the NTR in regulating both chaperone activity and assembly of a human sHSP. An evolutionary conserved region found in the middle of the NTR functions as both a negative regulator of activity and contains the necessary residues for interaction with another sHSP. Using the same set of technique, the effect of mutations in the ACD of HSPB1 that cause hereditary neuropathies was also investigated. The results described in Chapter 5 clearly show that most of these mutations increased the size of the protein and some, especially S135F had an increased chaperone activity. By investigating the same mutants using the ACD only constructs, we have shown that the effect of mutation does not lie within the ACD itself, as its structure was unaffected by mutation. The S135F however, had an unclear effect on the ACD structure as shown by small-angle X-ray scattering although the molecular mass still corresponded to an ACD dimer. Nonetheless, it thus seems that again the NTR is affected by these mutations as both the size and chaperone activity are regulated by sequences found in the N-terminal region. In summary, we have extensively characterized two human small heat shock proteins: HSPB1 and HSPB6. We have investigated the role of the NTR in defining two traits most commonly associated with sHSPs – chaperoning and assembly – and have found an essential role for a conserved region within the NTR. In an attempt to characterize disease-causing mutants of HSPB1 localized in the ACD, we found that although most did not have a profound effect on the ACD core structure, all full length proteins were larger and had a changed chaperoning profile against our standard substrate proteins. This suggests again an essential role for the NTR in disease pathology and we therefore suggest that future experiments should focus on the effect of mutation on the properties defined by the ACD-flanking regions. Furthermore, the techniques outlined in this thesis provide a useful toolbox for characterizing sHSPs and could serve as a guide for future experiments.nrpages: 187status: publishe

Everything but the ACD, functional conservation of the non-conserved regions in sHSPs

At the primary level small heat shock proteins are commonly described as a conserved α-crystallin domain flanked by regions that have disparate sequence content. While this holds true when analysing simple pairwise alignments, it belittles the importance of these N-terminal and C-terminal extensions. Careful examination of their sequences,combined with an improved understanding of the structure and activity of these proteins, yields an alternative view where the N- and C-terminal arms play an important role in function. In this chapter we shall describe the current understanding of these two regions and highlight that they both demonstrate structural and functional properties that are highly conserved across all kingdoms of life.status: accepte

Dissecting the Functional Role of the N-Terminal Domain of the Human Small Heat Shock Protein HSPB6

HSPB6 is a member of the human small heat shock protein (sHSP) family, a conserved group of molecular chaperones that bind partially unfolded proteins and prevent them from aggregating. In vertebrate sHSPs the poorly structured N-terminal domain has been implicated in both chaperone activity and the formation of higher-order oligomers. These two functionally important properties are likely intertwined at the sequence level, complicating attempts to delineate the regions that define them. Differing from the prototypical α-crystallins human HSPB6 has been shown to only form dimers in solution making it more amendable to explore the determinants of chaperoning activity alone. Using a systematic and iterative deletion strategy, we have extensively investigated the role of the N-terminal domain on the chaperone activity of this sHSP. As determined by size-exclusion chromatography and small-angle X-ray scattering, most mutants had a dimeric structure closely resembling that of wild-type HSPB6. The chaperone-like activity was tested using three different substrates, whereby no single truncation, except for complete removal of the N-terminal domain, showed full loss of activity, pointing to the presence of multiple sites for binding unfolding proteins. Intriguingly, we found that the stretch encompassing residues 31 to 35, which is nearly fully conserved across vertebrate sHSPs, acts as a negative regulator of activity, as its deletion greatly enhanced chaperoning capability. Further single point mutational analysis revealed an interplay between the highly conserved residues Q31 and F33 in fine-tuning its function.status: publishe

Dissecting the Functional Role of the N-Terminal Domain of the Human Small Heat Shock Protein HSPB6

<div><p>HSPB6 is a member of the human small heat shock protein (sHSP) family, a conserved group of molecular chaperones that bind partially unfolded proteins and prevent them from aggregating. In vertebrate sHSPs the poorly structured N-terminal domain has been implicated in both chaperone activity and the formation of higher-order oligomers. These two functionally important properties are likely intertwined at the sequence level, complicating attempts to delineate the regions that define them. Differing from the prototypical α-crystallins human HSPB6 has been shown to only form dimers in solution making it more amendable to explore the determinants of chaperoning activity alone. Using a systematic and iterative deletion strategy, we have extensively investigated the role of the N-terminal domain on the chaperone activity of this sHSP. As determined by size-exclusion chromatography and small-angle X-ray scattering, most mutants had a dimeric structure closely resembling that of wild-type HSPB6. The chaperone-like activity was tested using three different substrates, whereby no single truncation, except for complete removal of the N-terminal domain, showed full loss of activity, pointing to the presence of multiple sites for binding unfolding proteins. Intriguingly, we found that the stretch encompassing residues 31 to 35, which is nearly fully conserved across vertebrate sHSPs, acts as a negative regulator of activity, as its deletion greatly enhanced chaperoning capability. Further single point mutational analysis revealed an interplay between the highly conserved residues Q31 and F33 in fine-tuning its function.</p></div

N-terminal 10-residue deletion mutants.

<p>Cartoon of the different HSPB6 constructs used in this work. The α-crystallin domain (ACD) is shown in grey. The numbers at the top of the cartoon correspond to the residue number at domain boundaries.</p

Chaperone-like activity of HSPB6 and the 10-residue deletion mutants.

<p>Aggregation was monitored by following the absorbance at 340 nm for 90 min. The percentage protection for each construct was calculated as described in the Materials and Methods. (A) 0.25 mg/ml insulin used as a substrate. Aggregation was induced by adding a final concentration of 10 mM DTT prior to continuous measurement at 37°C. The ratios of substrate to sHSP are 1∶0.2 (black), 1∶0.1 (gray) and 1∶0.05 (light gray) (B) 0.25 mg/ml yADH used as a substrate. Aggregation was induced by adding 20 mM DTT and 2 mM EDTA (final concentration) prior to measurement at 42°C. The monomer mass molar ratios of substrate to sHSP were 1∶2 (black), 1∶1 (gray) and 1∶0.5 (light gray). (C) DTT-induced aggregation of HEWL in absence and presence of HSPB6 and the truncations. For clarity not all constructs are shown. The monomer mass molar ratio of HEWL:sHSP was 1∶2.</p

Size analysis by DLS of aggregated species.

a<p>HSPB6 was diluted in buffer to a concentration of 1 mg/ml and the measurement was performed at 35°C.</p>b<p>0.25 mg/ml of insulin was incubated with 10 mM DTT at 37°C prior to measurement to assure full aggregation.</p>c<p>0.25 mg/ml insulin was incubated with sHSP in the indicated monomer molar ratio to yield the same concentrations as used for the chaperone assays. This mixture was incubated with 10 mM DTT at 37°C prior to measurement.</p><p>Size analysis by DLS of aggregated species.</p

Solution properties of the HSPB6 10-residue deletion mutants.

<p>(A) Size-exclusion chromatography profiles of wild-type HSPB6, Δ31–40 and Δ61–70. 100 µl of protein at three different concentrations was loaded on a Superdex 200 10/300 GL column. 2.5 mg/ml is depicted as a solid line, 5 mg/ml as a dashed line and 10 mg/ml as a dotted line. The absorbance was normalized to the maximum absorbance for each curve. (B) Bis-ANS titration curves for HSPB1, HSPB6 and HSPB4. 2.5 µM protein was mixed with increasing concentrations of bis-ANS and fluorescence intensity was recorded at 490 nm using an excitation wavelength of 390 nm. HSPB6 is shown in yellow, HSPB1 in black and HSPB4 in grey. (C) Bis-ANS titration curves for all constructs, the same setup as for HSPB1 and HSPB4 was used. HSPB6 is shown in yellow, ΔN11 in blue, Δ11–20 in cyan, Δ21–30 in red, Δ31–40 in green, Δ41–50 in orange, Δ51–60 in pink and Δ61–70 in violet.</p