International Journal of Molecular Sciences

Article The Heterooligomerization of Human Small Heat Shock Is Controlled by Conserved Motif Located in the N-Terminal Domain

Vladislav M. Shatov 1, Sergei V. Strelkov 2 and Nikolai B. Gusev 1,* 1 Department of Biochemistry, School of Biology, Moscow State University, Moscow 119991, Russian; [email protected] 2 Laboratory for Biocrystallography, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, 3000 Leuven, Belgium; [email protected] * Correspondence: [email protected]

 Received: 19 May 2020; Accepted: 12 June 2020; Published: 15 June 2020 

Abstract: Ubiquitously expressed human small heat shock proteins (sHsps) HspB1, HspB5, HspB6 and HspB8 contain a conserved motif (S/G)RLFD in their N-terminal domain. For each of them, we prepared mutants with a replacement of the conserved R by A (R/A mutants) and a complete deletion of the pentapeptide (∆ mutants) and analyzed their heterooligomerization with other wild-type (WT) human sHsps. We found that WT HspB1 and HspB5 formed heterooligomers with HspB6 only upon heating. In contrast, both HspB1 mutants interacted with WT HspB6 even at low temperature. HspB1/HspB6 heterooligomers revealed a broad size distribution with equimolar ratio suggestive of heterodimers as building blocks, while HspB5/HspB6 heterooligomers had an approximate 2:1 ratio. In contrast, R/A or ∆ mutants of HspB6, when mixed with either HspB1 or HspB5, resulted in heterooligomers with a highly variable molar ratio and a decreased HspB6 incorporation. No heterooligomerization of HspB8 or its mutants with either HspB1 or HspB5 could be detected. Finally, R/A or ∆ mutations had no effect on heterooligomerization of HspB1 and HspB5 as analyzed by ion exchange chromatography. We conclude that the conserved N-terminal motif plays an important role in heterooligomer formation, as especially pronounced in HspB6 lacking the C-terminal IXI motif.

Keywords: small heat shock proteins; HspB1; HspB5; HspB6; HspB8; heterooligomerization

1. Introduction Small heat shock proteins (sHsp) form a large family of ubiquitously expressed proteins playing an important role in proteostasis [1–3]. Human genomes contains ten encoding sHsps [4,5] and four members (HspB1, HspB5, HspB6 and HspB8) of this large family are expressed in practically all human tissues [6,7]. In certain tissues (for instance, in skeletal muscles and heart), some of these proteins are simultaneously expressed at comparably high level, reaching up to 3–5% of the total soluble proteins [8]. Small heat shock proteins tend to form oligomers of various size [9,10]. In line with a considerable conservation of primary structure across different sHsps [11], they are capable of forming heterooligomeric complexes [12–14] which are structurally and functionally distinct from the corresponding homooligomers. The formation of heterooligomeric complexes formed by HspB1 and HspB5 [14,15], HspB5 and HspB6 [14,16–18], HspB8 and other sHsps [19,20] has been reported in the literature. The interaction of HspB1 and HspB6 and their ability to form heterooligomeric complexes has been analyzed especially rigorously [21–24]. Special attempts were undertaken in order to establish the formation of heterooligomeric complexes among all four ubiquitously expressed sHsps (HspB1, HspB5, HspB6, HspB8) [19,25,26].

Int. J. Mol. Sci. 2020, 21, 4248; doi:10.3390/ijms21124248 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 2 of 20 Int. J. Mol. Sci. 2020, 21, 4248 2 of 18 order to establish the formation of heterooligomeric complexes among all four ubiquitously expressed sHsps (HspB1, HspB5, HspB6, HspB8) [19,25,26]. ItIt is is well-accepted well-accepted that that all all parts parts of sHsps,of sHsps, i.e., i.e., N-terminal N-terminal (NTD), (NTD),α-crystallin α-crystallin (ACD) (ACD) and C-terminaland C- domainsterminal (CTD) domains can play (CTD) significant can play role significant in the formation role in of the heterooligomeric formation of heterooligomeric complexes [18]. The complexesα-crystallin domain[18]. The of sHsps α-crystallin is highly domain conserved of [sHsps11] and is therefore highly cannotconserved determine [11] and specificity therefore of heterooligomerization.cannot determine Atspecificity the same of time, heterooligomerization. it is known that the At interaction the same ti betweenme, it is sHspsknown isthat highly the interaction specific and between depends sHsps on the natureis highly of proteinspecific partnersand depends [13, 25on, 26the]. nature Human of sHspsprotein differpartners in [13,25,26]. the presence Human or absence sHsps differ of conserved in the IX(Ipresence/V) tripeptide or absence located of conserved in the C-terminal IX(I/V) tripeptide domain possessinglocated in the high C-terminal mobility domain and involved possessing in formation high ofmobility intra- and and inter-subunit involved in formation contacts of in intra- large and oligomeric inter-subunit complexes contacts [27 in]. large In addition, oligomeric sHsps complexes differ in the[27]. size In and addition, composition sHsps ofdiffer their in N-terminal the size and domains composition playing of important their N-terminal role in stabilizationdomains playing of large homooligomersimportant role [ 9,in28 –stabilization31]. Therefore, of thelarge specificity homoolig ofomers heterooligomerization [9,28–31]. Therefore, seems the to be specificity predominantly of dependentheterooligomerization on the structure seems and to composition be predominantly of variable dependent C- and N-terminal on the structure domains and of composition sHsps. of variableUbiquitously C- and N-terminal expressed domains human of sHsps sHsps. (HspB1, HspB5, HspB6 and HspB8) differ in the size and structureUbiquitously of their expressed N-terminal human domains. sHsps (HspB1, The latterHspB5, play HspB6 an and important HspB8) rolediffer in in chaperone-likethe size and activitystructure [30 ,of32 ,their33], inN-terminal formation domains. of homooligomers The latter [pl9,ay10 ,29an, 31important,32] and role in formation in chaperone-like of heterooligomeric activity [30,32,33], in formation of homooligomers [9,10,29,31,32] and in formation of heterooligomeric complexes [18,23,24]. Although the length and the primary structure of the N-terminal domain of complexes [18,23,24]. Although the length and the primary structure of the N-terminal domain of sHsps is highly variable [11], all these proteins contain a conserved (S/G)RLFD(D/Q)xFG motif located sHsps is highly variable [11], all these proteins contain a conserved (S/G)RLFD(D/Q)xFG motif at the beginning of this domain (Figure1). In order to analyze the role of this motif in the process of located at the beginning of this domain (Figure 1). In order to analyze the role of this motif in the heterooligomerization, we replaced highly conserved Arg in this sequence with Ala (R/A mutants) process of heterooligomerization, we replaced highly conserved Arg in this sequence with Ala (R/A or completely deleted the first five residues of this motif (∆ mutants) and compared the pairwise mutants) or completely deleted the first five residues of this motif (Δ mutants) and compared the heterooligomerizationpairwise heterooligomerization of the wild-type of the (WT)wild-type proteins (WT) and proteins their Rand/A andtheir∆ R/Amutants and Δ by mutants means ofby size exclusionmeans of or size ion-exchange exclusion or chromatography. ion-exchange chromatography.

FigureFigure 1. 1.Alignment Alignment ofof thethe primaryprimary structure of of the the N-terminal N-terminal domains domains (NTD) (NTD) of offour four human human small small heatheat shock shock proteins proteins (sHsp). (sHsp). TheThe conserved (S/G)RLFD(D/Q)xFG (S/G)RLFD(D/Q)xFG motif motif is ismarked marked grey, grey, the the conserved conserved pentapeptidepentapeptide is is depicted depicted inin boldbold letters, the co conservednserved Arg Arg residue residue is ismarked marked red. red. The The following following sequencessequences from from Uniprot Uniprot were were used: used: P04792 P04792 (HspB1), (HspB1), P02511 P02511 (HspB5), (HspB5), O14558 O14558 (HspB6), (HspB6), Q9UJY1 Q9UJY1 (HspB8). Multiple(HspB8). alignment Multiple alignment was generated was generated by Tcoffee. by Tcoffee.

2.2. Results Results

2.1.2.1. Characterization Characterization of of AnalyzedAnalyzed ProteinsProteins TheThe method method of of expression expression andand purificationpurification prov providedided highly highly purified purified samples samples of ofthe the WT WT sHsps sHsps andand their their R/ AR/A and and∆ mutants Δ mutants (Figure (Figure2). Point 2). mutation Point mutation R /A had R/A no noticeable had no enoticeableffect on electrophoretic effect on mobility,electrophoretic whereas mobility, the deletion whereas of pentapeptide the deletion wasof pentapeptide accompanied was by accompanied a small increase by a ofsmall electrophoretic increase mobilityof electrophoretic of all proteins mobility on sodium of all proteins dodecyl on sulfatesodium (SDS) dodecyl gel sulfat electrophoresise (SDS) gel electrophoresis (Figure2). (Figure 2). 2.2. Effect of R/A and ∆ Mutations on the Interaction of HspB1 and HspB6 The WT HspB1 forms large oligomers with an apparent molecular mass of 530-600 kDa [30,34]. As reported earlier [35], the R/A mutation resulted in the partial dissociation of large oligomers of HspB1 and deletion of the conserved pentapeptide resulted in complete dissociation of large HspB1 oligomers and accumulation of small oligomers with apparent molecular weight of 90 kDa (Figure3). The R /A or ∆ mutations had no effect on the quaternary structure of HspB6, and both the WT and its R/A and ∆ mutants formed only small oligomers with an apparent molecular weight of 58–60 kDa on the size exclusion chromatography (SEC).

Int. J. Mol. Sci. 2020, 21, 4248 3 of 18 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 20

Figure 2. Sodium dodecyl sulfate (SDS) gel electrophoresi electrophoresiss of the final final preparation of the wild-type (WT) proteins and their mutants. About 1.5 µgµg of proteins were loaded on each track.track.

2.2. EffectIn line of withR/A and previous Δ Mutations observations on the [Interaction21,22], mixing of HspB1 equimolar and HspB6 amounts of WT HspB1 and HspB6 at 42 C resulted in the formation of a broad spectrum of heterooligomeric complexes in a SEC experiment, ◦ The WT HspB1 forms large oligomers with an apparent molecular mass of 530-600 kDa [30,34]. with apparent molecular weight peaking at about 100 and 300 kDa (Figure3). The stoichiometry of As reported earlier [35], the R/A mutation resulted in the partial dissociation of large oligomers of HspB1/HspB6 was close to equimolar in all chromatographic fractions. Towards further quantification, HspB1 and deletion of the conserved pentapeptide resulted in complete dissociation of large HspB1 here we have introduced a correlation coefficient based on the presence of either partner across the oligomers and accumulation of small oligomers with apparent molecular weight of 90 kDa (Figure chromatographic fractions (see Methods). Such a correlation coefficient is a convenient integral measure 3). The R/A or Δ mutations had no effect on the quaternary structure of HspB6, and both the WT which reflects the propensity of a given pair of sHsps to form heterocomplexes. For heated WT HspB1 protein and its R/A and Δ mutants formed only small oligomers with an apparent molecular weight and HspB6 samples, the correlation coefficient was close to 1, indicating a strong preference towards of 58–60 kDa on the size exclusion chromatography (SEC). heterooligomerization consistent with previous reports [24]. In contrast, unheated mix of HspB1 and HspB6 resulted in a correlation coefficient close to 0. This reflects the fact that the two proteins do not interact and elute in very different parts of the chromatogram, namely as large HspB1 homooligomers and much smaller HspB6 homodimers (Figure3). When the mixture of R/A mutant of HspB1 and the WT HspB6 preincubated at low temperature was loaded on the column, the HspB1 mutant was detected in practically all fractions of the elution profile, including the fractions 34–35 containing proteins with an apparent molecular weight of 58–60 kDa (Figure3B). The molar ratio HspB1 /HspB6 in these complexes varied in the range of 5:1 to 1:1 (Table1). This means that even at a low temperature, the R /A mutant of HspB1 can interact and form heterooligomers with the WT HspB6 with variable stoichiometry. At a high temperature, this mutant formed three different heterooligomeric complexes with the WT HspB6 with apparent molecular weights of 100, 210 and 300 kDa. Stoichiometry HspB1/HspB6 in all these complexes was close to equimolar. After the mixture of ∆ mutant of HspB1 and WT HspB6 preincubated at low temperature had been loaded on the column, we detected a decrease in the peak corresponding to isolated HspB6 (fractions 33–35) and appearance of HspB6 in fractions 21–27 where it was eluted together with ∆ mutant of HspB1 (Figure3C). This was apparent in a correlation coe fficient of 0.82 between the elution profiles for both components. This means that even at low temperature ∆ mutant of HspB1 forms heterooligomeric complexes with the WT HspB6 with variable stoichiometry (Table1). When heated mixture of ∆ mutant of HspB1 and WT HspB6 was loaded on the column, we detected two peaks of heterooligomers with apparent molecular weight of 100 and 260 kDa, where stoichiometry HspB1/HspB6 was constant and close to 1:1 (Figure3C). Interestingly, the WT HspB6 was able to promote re-association of ∆ mutant of HspB1 and formation of heterooligomeric complexes with high molecular weight (fractions 23–26 on Figure3C).

Int. J. Mol. Sci. 2020, 21, 4248 4 of 18

FigureFigure 3. 3.Size-exclusion Size-exclusion chromatography chromatography (SEC) of the of the WT WT HspB1 HspB1 (A), (A its), andR/A its(B) R /andA(B Δ) and(C) mutants∆ (C) and their mutantsmixtures and with their the mixtures WT HspB6. with the Elution WT HspB6. profiles Elution of isolated profiles HspB6 of isolated is shown HspB6 as are black shown dotted as a line, that of blackisolated dotted HspB1 line; thator its of mutants isolatedHspB1 by black or itsdashed mutants line. by Elution black dashed profileline. of the The mixture elution profileof HspB1 of species and theHspB6 mixture preincubated of HspB1 species at low and temperature HspB6 preincubated is shown at low as solidtemperature blue line is shown and that as a solid of the blue corresponding linemixture and that preincubated of the corresponding at elevated mixture temperature preincubated is shown at elevated as solid temperature red line. Molar is shown ratio asHspB1/HspB6 solid (grey redsolid line. line) The molarin corresponding ratio HspB1/ HspB6fractions (grey obtained solid line) after in the chromatography corresponding fractionsof preheated obtained sample after is indicated chromatographyon the right ordinate. of preheated Protein sample composition is indicated of onfractions the right obtained ordinate. after The proteinchromatography composition of of HspB1/HspB6 themixture fractions preincubated obtained after at chromatography low (labeled blue) of HspB1 or high/HspB6 (labeled mixture red) preincubated temperature at are low presented (labeled on inserts. blue)Correlation or high (labeled coefficient red) temperature (Corr) is indicated are presented under on eachinserts. electrophoregram. Correlation coefficient Numbers (Corr) is above tracks indicated under each electropherogram. The numbers above tracks correspond to the fractions number. correspond to the fractions number. Apparent molecular weights are indicated by arrows. Apparent molecular weights are indicated by arrows. Int. J. Mol. Sci. 2020, 21, 4248 5 of 18

Table 1. Correlation coefficient and molar ratio HspB1/HspB5, HspB1/HspB6 and HspB5/HspB6 in heterooligomers (in brackets).

WT HspB1 R/A HspB1 ∆ HspB1 WT HspB5 R/A HspB5 ∆ HspB5 Without heating 0 0 0 WT HspB5 After heating 1.00 (constant ~1.0) 1.00 (constant ~1.0) 1.00 (constant ~1.0) Without heating 0 R/A HspB5 After heating 1.00 (constant ~1.0) Without heating 0 ∆ HspB5 After heating 1.00 (constant ~1.0) Without heating 0.05 0.31 (variable 5–1) 0.82 (variable 3–1) 0.05 0.12 0 WT HspB6 After heating 0.99 (constant ~1.1) 0.99 (constant ~1.1) 1.00 (constant ~1.2) 0.99 (constant ~2.0) 0.99 (variable 3–2) 0.98 (variable 5–2) Without heating 0.04 0.12 R/A HspB6 After heating 0.78 (variable 10–1) 0.99 (variable 4–1) Without heating 0.05 0 ∆ HspB6 After heating 0.32 (variable 10–1) 0.98 (variable 4–1) Without heating 0 0 0 0 0 0 WT HspB8 After heating 0 0 0 0 0 0 Without heating 0 0 R/A HspB8 After heating 0 0 Without heating 0 0 ∆ HspB8 After heating 0 0 Correlation coefficient was calculated as described in the Materials and Methods section. If the correlation coefficient was larger than 0.30, we calculated the molar ratio HspB1/HspB5, HspB1/HspB6 and HspB5/HspB6 in different fractions of size-exclusion chromatography (Figures3–8). Variations of this molar ratio are indicated in brackets. Values deviating from the WT case are in bold.

At a low temperature, the R/A mutant of HspB6 was unable to form heterooligomeric complexes with the WT HspB1 (Figure4B). Next, a mixture of R /A mutant of HspB6 and the WT HspB1 was heated to 42 ◦C prior to chromatography. The WT HspB1 was detected in practically all fractions and the stoichiometry HspB1/HspB6 varied between 10:1 and 1:1 (Table1). The only one pronounced peak with apparent molecular weight of ~100 kDa (fractions 29–31 in Figure4B) contained a constant ratio of HspB1 and R/A mutant of HspB6 that was close to 2:1. At low temperature ∆ mutant of HspB6 was also unable to form heterooligomeric complexes with the WT HspB1 (Figure4C). At high temperatures, ∆ mutant of HspB6 induced partial dissociation of large oligomers of HspB1 and therefore HspB1 was distributed over the whole elution profile (Figure4C) without being able to form any complexes with fixed stoichiometry with the ∆ mutant of HspB6 (Table1).

2.3. Effect of R/A and ∆ Mutations on the Interaction of HspB5 and HspB6 The WT HspB5 and its R/A or ∆ mutants formed large stable oligomers with apparent molecular weights of 510–550 kDa [35]. At low temperature the WT HspB5 was unable to form heterooligomeric complexes with the WT HspB6 and each protein was eluted in separate peak with apparent molecular weight of 540–550 and 58–60 kDa (Figure5A). After heating, the WT HspB5 and the WT HspB6 formed one type of heterooligomers with an apparent molecular weight of 400 kDa (Figure5A). The stoichiometry of HspB5/HspB6 in this complex was close to 2:1 (Table1) and about half of HspB6 remained unbound and was not included in heterooligomers. The R/A mutant of HspB5 formed heterooligomers with the WT HspB6 only at elevated temperature. These complexes had apparent molecular weight of 350 kDa and the stoichiometry HspB5/HspB6 varied in the range of 3:1 to 2:1 (Table1). Under these conditions, a larger portion of HspB6 remained free and was not included in heterooligomeric complex. Similar results were obtained with ∆ mutant of HspB5. Indeed, heterooligomeric complexes were formed only at elevated temperature, had an apparent molecular weight of ~350 kDa and stoichiometry HspB5/HspB6 varying in the range 5:1 and 2:1 (Figure5C, Table1). At low temperature neither R/A nor ∆ mutants of HspB6 were able to form heterooligomeric complexes with the WT HspB5 (Figure6B,C). At high temperature these mutants of HspB6 formed one type of heterooligomers with HspB5 with apparent molecular weight of 400 kDa. The stoichiometry Int. J. Mol. Sci. 2020, 21, 4248 6 of 18

HspB5/HspB6 in this heterogeneous complex was variable (Table1) and significant portion of HspB6 mutants remained free and were not included in heterooligomeric complex (Figure6B,C). Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 20

FigureFigure 4. Size-exclusion 4. Size-exclusion chromatography chromatography of the WT WT HspB6 HspB6 (A) (A, ),its its R/A R /(B)A( Band) and Δ (C)∆ (mutantsC) mutants and and theirtheir mixtures mixtures with with the the WT WT HspB1. HspB1. The The elution elution profilesprofiles of of isolated isolated HspB6 HspB6 or its or mutants its mutants are shown are shown as a blackas a black dotted dotted line, line, that that of of isolated isolated WT WT HspB1HspB1 by a a black black dashed dashed line. line. The The elution elution profile profile of the of the mixture of HspB6 species and WT HspB1 preincubated at low temperature is shown as a solid blue mixture of HspB6 species and WT HspB1 preincubated at low temperature is shown as a solid blue line and that of the corresponding mixture preincubated at elevated temperature is shown as a solid line and that of the corresponding mixture preincubated at elevated temperature is shown as a solid red line. The molar ratio HspB1/HspB6 (grey solid line) in the corresponding fractions obtained after red line.chromatography The molar ratio of preheated HspB1/ HspB6sample (greyis indicated solid on line) the in right the ordinate. corresponding The protein fractions composition obtained of after chromatographythe fractions ofobtained preheated after sample the chromatography is indicated on of the HspB1/HspB6 right ordinate. mixture The protein preincubated composition at low of the fractions(labeled obtained blue) afteror high the (labeled chromatography red) temperatur of HspB1e are/ HspB6presented mixture on the preincubated inserts. The atcorrelation low (labeled blue)coefficient or high (labeled (Corr) is red) indicated temperature under each are electr presentedopherogram. on the The inserts. numbers The correlationabove tracks coe correspondfficient (Corr) to is indicatedthe fractions under eachnumber. electropherogram. Apparent molecu Thelar weights numbers are above indicated tracks by correspondarrows. to the fractions number. Apparent molecular weights are indicated by arrows. 2.3. Effect of R/A and Δ Mutations on the Interaction of HspB5 and HspB6 The WT HspB5 and its R/A or Δ mutants formed large stable oligomers with apparent molecular weights of 510–550 kDa [35]. At low temperature the WT HspB5 was unable to form heterooligomeric

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 8 of 20

complexes with the WT HspB6 and each protein was eluted in separate peak with apparent molecular weight of 540–550 and 58–60 kDa (Figure 5A). After heating, the WT HspB5 and the WT HspB6 formed one type of heterooligomers with an apparent molecular weight of 400 kDa (Figure 5A). The stoichiometry of HspB5/HspB6 in this complex was close to 2:1 (Table 1) and about half of HspB6 remained unbound and was not included in heterooligomers. The R/A mutant of HspB5 formed heterooligomers with the WT HspB6 only at elevated temperature. These complexes had apparent molecular weight of 350 kDa and the stoichiometry HspB5/HspB6 varied in the range of 3:1 to 2:1 (Table 1). Under these conditions, a larger portion of HspB6 remained free and was not included in heterooligomeric complex. Similar results were obtained with Δ mutant of HspB5. Indeed, heterooligomeric complexes were formed only at elevated temperature, had an apparent molecular Int. J. Mol. Sci. 2020, 21, 4248 7 of 18 weight of ~350 kDa and stoichiometry HspB5/HspB6 varying in the range 5:1 and 2:1 (Figure 5C, Table 1).

Figure 5. Size-exclusion chromatography of the WT HspB5 (A), its R/A(B) and ∆ (C) mutants and their Figure 5. Size-exclusion chromatography of the WT HspB5 (A), its R/A (B) and Δ (C) mutants and mixtures with the WT HspB6. The elution profiles of isolated HspB6 are shown as a black dotted line, their mixtures with the WT HspB6. The elution profiles of isolated HspB6 are shown as a black dotted that of isolated WT HspB5 or its mutants by a black dashed line. The elution profile of the mixture of line, that of isolated WT HspB5 or its mutants by a black dashed line. The elution profile of the mixture HspB5 species and WT HspB6 preincubated at low temperature is shown as a solid blue line and that of of HspB5 species and WT HspB6 preincubated at low temperature is shown as a solid blue line and the corresponding mixture preincubated at elevated temperature is shown as a solid red line. The molar that of the corresponding mixture preincubated at elevated temperature is shown as a solid red line. ratio HspB5/HspB6 (grey solid line) in the corresponding fractions obtained after chromatography of preheated sample is indicated on the right ordinate. The protein composition of the fractions obtained after the chromatography of HspB5/HspB6 mixture preincubated at low (labeled blue) or high (labeled red) temperature are presented on inserts. The correlation coefficient (Corr) is indicated under each electropherogram. The numbers above tracks correspond to the fractions number. Apparent molecular weights are indicated by arrows. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 20

The molar ratio HspB5/HspB6 (grey solid line) in the corresponding fractions obtained after chromatography of preheated sample is indicated on the right ordinate. The protein composition of the fractions obtained after the chromatography of HspB5/HspB6 mixture preincubated at low (labeled blue) or high (labeled red) temperature are presented on inserts. The correlation coefficient (Corr) is indicated under each electropherogram. The numbers above tracks correspond to the fractions number. Apparent molecular weights are indicated by arrows.

At low temperature neither R/A nor Δ mutants of HspB6 were able to form heterooligomeric complexes with the WT HspB5 (Figure 6B,C). At high temperature these mutants of HspB6 formed one type of heterooligomers with HspB5 with apparent molecular weight of 400 kDa. The stoichiometry HspB5/HspB6 in this heterogeneous complex was variable (Table 1) and significant Int. J. Mol. Sci. 2020, 21, 4248 8 of 18 portion of HspB6 mutants remained free and were not included in heterooligomeric complex (Figure 6B,C).

Figure 6. Size-exclusion chromatography of the WT HspB6 (A), its R/A(B) and ∆ (C) mutants and their Figure 6. Size-exclusion chromatography of the WT HspB6 (A), its R/A (B) and Δ (C) mutants and mixtures with the WT HspB5. The elution profiles of isolated HspB6 or its mutants are shown as a black their mixtures with the WT HspB5. The elution profiles of isolated HspB6 or its mutants are shown dotted line, that of isolated WT HspB5 by a black dashed line. The elution profile of the mixture of HspB6 species and WT HspB5 preincubated at low temperature is shown as a solid blue line and that of

the corresponding mixture preincubated at elevated temperature is shown as a solid red line. The molar ratio HspB5/HspB6 (grey solid line) in the corresponding fractions obtained after the chromatography of preheated sample is indicated on the right ordinate. Protein compositions of fractions obtained after chromatography of HspB5/HspB6 mixture preincubated at low (labeled blue) or high (labeled red) temperature are presented on inserts. The correlation coefficient (Corr) is indicated under each electropherogram. The numbers above tracks correspond to the fractions number. Apparent molecular weights are indicated by arrows. Int. J. Mol. Sci. 2020, 21, 4248 9 of 18 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 11 of 20

Figure 7. Size exclusion chromatography of the WT HspB1 and the WT HspB8 (A) or its R/A(B) or Figure 7. Size exclusion chromatography of the WT HspB1 and the WT HspB8 (A) or its R/A (B) or Δ ∆ (C) mutants. The elution profile of HspB8 and its mutants is shown as a black dotted line and that (C) mutants. The elution profile of HspB8 and its mutants is shown as a black dotted line and that of of isolated HspB1 as a black dashed line. The elution profile of the mixture of HspB1 and HspB8 isolated HspB1 as a black dashed line. The elution profile of the mixture of HspB1 and HspB8 species species preincubated at elevated temperature is shown as a solid red line. The protein compositions of preincubated at elevated temperature is shown as a solid red line. The protein compositions of fractions obtained after chromatography of HspB1/HspB8 mixture preincubated at high temperature are fractions obtained after chromatography of HspB1/HspB8 mixture preincubated at high temperature presented on inserts. The numbers above tracks correspond to the fractions number. Apparent molecular are presented on inserts. The numbers above tracks correspond to the fractions number. Apparent weights are indicated by arrows. molecular weights are indicated by arrows.

2.5. Formation of Heterooligomeric Complexes between HspB1 and HspB5 Both HspB1 and HspB5 form similar size large homooligomers, and therefore the formation of heterooligomeric complexes of these proteins cannot be followed by means of size-exclusion chromatography. At the same time, these proteins have different isoelectric points, and therefore can be separated by means of ion-exchange chromatography. At pH 6.8, isolated HspB5 with a theoretical

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 12 of 20

isoelectric point 6.76 was not retarded on the DEAE HiTrap column, whereas HspB1 with a theoretical isoelectric point 5.98 was tightly bound to the column and was eluted only by salt gradient (Figure 8). When the mixture of the WT HspB1 and HspB5 preincubated at low temperature was loaded on the DEAE HiTrap column, HspB5 was not retarded on the column and was eluted in the flow through, whereas HspB1 was retarded on the column and eluted by salt gradient in a position corresponding to the isolated protein (Figure 8). Only one peak was detected on the elution profile of ion exchange chromatography when a preheated mixture of HspB1 and HspB5 was loaded on the DEAE HiTrap column (Figure 8). This peak was less tightly bound to the column than isolated HspB1 and contained equimolar mixture of HspB1 and HspB5 (Figure 8). We have run similar experiments with different mixtures containing R/A or Δ mutants of HspB1 and/or HspB5 and have not detected Int. J.any Mol. differences Sci. 2020, 21, 4248in behavior of the WT proteins and their mutants. Thus, mutations in the N-terminal10 of 18 domain negligibly affect interaction of HspB1 and HspB5.

FigureFigure 8. Ion-exchange 8. Ion-exchange chromatography chromatography of the of WTthe WT HspB1 HspB1 and and HspB5. HspB5. The The elution elution profile profile of isolated of isolated HspB1HspB1 and and HspB5 HspB5 are are shown shown as blackas black dashed dashed and and dotted dotted lines, lines, respectively. respectively. The The elution elution profile profile ofof the the mixture of HspB1 HspB1 and and HspB5 HspB5 preincubated preincubated at atlow low temp temperatureerature is shown is shown as a as solid a solid blue blueline and line that andof that the of corresponding the corresponding mixture mixture preincubated preincubated at elevat ated elevated temperature temperature is shown is as shown a solid as red a solidline. The red line.molar The ratio molar HspB1/HspB5 ratio HspB1 (grey/HspB5 solid (greyline) in solid corresp line)onding in corresponding fractions obtained fractions after obtained chromatography after chromatographyof preheated ofsample preheated is indicated sample on is indicatedthe right ordi on thenate. right Conductivity ordinate. Conductivity is shown as a is solid shown black as a line. solidThe black protein line. The compositions protein compositions of fractions of fractionsobtained obtained after chromatography after chromatography of HspB1/HspB5 of HspB1/HspB5 mixture mixturepreincubated preincubated at low at low (labeled (labeled blue) blue) or or high high (lab (labeledeled red)red) temperaturetemperature are are presented presented on inserts.on inserts. CorrelationCorrelation coe fficoefficientcient (Corr) (Corr) is indicated is indicated under under each each electropherogram. electropherogram. The The numbers numbers above above tracks tracks correspondcorrespond to the to fractionsthe fractions number. number.

2.4.3. Lack Discussion of Interaction of HspB8 and Its Mutants with HspB1 and HspB5 WhenThe the heterooligomerization mixture of the WT HspB1 of various and the human WT HspB8 sHsps preincubated has been frequently at high temperature reported [14,25,26], was loadedalthough on the the Superdex physiological 200 column, implications we detected of this two process separate are peaks still poorly with apparent understood. molecular The experiments weights of 590reported and 35 here kDa, indicate each of an which important contained involvement isolated HspB1 of theand conserved HspB8, pentapeptide respectively (Figurewithin7 A).the N- Similarterminal results motif were of obtained selected inhuman the case sHsps of the on mixturetheir heterooligomer containing R/izationA mutant properties. of HspB8 and the WT HspB1 (FigureAlthough7B), the except whole for thepeptide fact that,(S/G)RLFD as expected, in the the N- apparentterminal moleculardomain is weightconserved, of the the R /singleA mutantpositively of HspB8 charged was larger Arg residue than that is ofespecially the WT proteinpresented and in was practically close to 65all kDavertebrate [35]. The sHsp∆ mutant homologues of HspB8(HspB1, was alsoHspB2, unable HspB4, to interact HspB5, with HspB6 the WT and HspB1 HspB8) and [30]. two Point proteins mutation again R/A were decreases eluted in separatethe positive peakscharge (Figure in 7theC). N-terminal Qualitatively part similar of HspB1 results and were results obtained in a partial with dissociation the mixture ofof thelarge WT HspB1 HspB8 oligomers and the(Figure WT HspB5 3). In or this its Rrespect,/A or ∆ themutants. R/A replacement Thus, even isat similar elevated to that temperature induced by HspB8 Ser15 was phosphorylation unable to interactleading with to HspB1 decrease or HspB5. of positive and increase of negative charge of the N-terminal domain and provoking dissociation of HspB1 [34,36]. We can speculate that the decrease of the positive (or 2.5. Formation of Heterooligomeric Complexes between HspB1 and HspB5 increase of negative) charge of the N-terminal domain induces dissociation of large HspB1 oligomers. InBoth line HspB1with previous and HspB5 observations form similar [23,24], size the large R/A homooligomers, mutant of HspB1 and formed therefore heterooligomers the formation with of heterooligomeric complexes of these proteins cannot be followed by means of size-exclusion chromatography. At the same time, these proteins have different isoelectric points, and therefore can be separated by means of ion-exchange chromatography. At pH 6.8, isolated HspB5 with a theoretical isoelectric point 6.76 was not retarded on the DEAE HiTrap column, whereas HspB1 with a theoretical isoelectric point 5.98 was tightly bound to the column and was eluted only by salt gradient (Figure8). When the mixture of the WT HspB1 and HspB5 preincubated at low temperature was loaded on the DEAE HiTrap column, HspB5 was not retarded on the column and was eluted in the flow through, whereas HspB1 was retarded on the column and eluted by salt gradient in a position corresponding to the isolated protein (Figure8). Only one peak was detected on the elution profile of ion exchange chromatography when a preheated mixture of HspB1 and HspB5 was loaded on the DEAE HiTrap Int. J. Mol. Sci. 2020, 21, 4248 11 of 18 column (Figure8). This peak was less tightly bound to the column than isolated HspB1 and contained equimolar mixture of HspB1 and HspB5 (Figure8). We have run similar experiments with di fferent mixtures containing R/A or ∆ mutants of HspB1 and/or HspB5 and have not detected any differences in behavior of the WT proteins and their mutants. Thus, mutations in the N-terminal domain negligibly affect interaction of HspB1 and HspB5.

3. Discussion The heterooligomerization of various human sHsps has been frequently reported [14,25,26], although the physiological implications of this process are still poorly understood. The experiments reported here indicate an important involvement of the conserved pentapeptide within the N-terminal motif of selected human sHsps on their heterooligomerization properties. Although the whole peptide (S/G)RLFD in the N-terminal domain is conserved, the single positively charged Arg residue is especially presented in practically all vertebrate sHsp homologues (HspB1, HspB2, HspB4, HspB5, HspB6 and HspB8) [30]. Point mutation R/A decreases the positive charge in the N-terminal part of HspB1 and results in a partial dissociation of large HspB1 oligomers (Figure3). In this respect, the R /A replacement is similar to that induced by Ser15 phosphorylation leading to decrease of positive and increase of negative charge of the N-terminal domain and provoking dissociation of HspB1 [34,36]. We can speculate that the decrease of the positive (or increase of negative) charge of the N-terminal domain induces dissociation of large HspB1 oligomers. In line with previous observations [23,24], the R/A mutant of HspB1 formed heterooligomers with the WT HspB6 even at low temperature that is unusual for the WT proteins (Table1). This can be due to more e ffective heterooligomerization of already partially dissociated species of HspB1. At high temperatures, the R/A mutant of HspB1 formed different heterooligomers with HspB6, with apparent molecular weights of 100, 210 and 300 kDa, each of which contained equimolar quantities of both sHsps (Figure3B, Table1). The deletion of the pentapeptide SRLFD induced a dramatic destabilization of HspB1 homooligomers and the ∆ mutant of HspB1 was presented in the form of heterogeneous small oligomers (Figure3C). Even at low temperature, these small oligomers of the HspB1 ∆ mutant formed a heterogeneous mixture of heterooligomers with the WT HspB6, with variable stoichiometry and correlation coefficient close to 0.82 (Table1). At elevated temperatures, the ∆ mutant of HspB1 formed two types of heterooligomeric complexes with HspB6, with apparent molecular weights of 100 and 260 kDa with fixed stoichiometry close to 1:1, and practically indistinguishable from those formed by the WT HspB1 (Figure3C, Table1). It is remarkable that under these conditions HspB6 was able to reconstitute large oligomers that were completely absent in the case of the isolated ∆ mutant of HspB1. Neither the R/A mutation [35] nor the deletion of the conserved peptide affected the quaternary structure of HspB6, and both the WT protein and its mutants were eluted on size exclusion chromatography in the peak, with an apparent molecular weight of 58–60 kDa (Figure4). These data agree with the earlier published results of Heirbaut et al. [24,33]. Heterooligomers formed by the R/A mutant of HspB6 and the WT HspB1 at high temperatures were very heterogeneous. Stoichiometry HspB1/HspB6 in these complexes was variable, and, in the most prominent heterocomplex, with an apparent molecular weight of 100 kDa, was close to 2:1 (Figure4B, Table1). After preincubation at elevated temperatures, the ∆ mutant of HspB6 induced partial dissociation of large oligomers of HspB1 and different oligomers species of HspB1 were distributed over the whole elution profile without forming a separate peak of heterooligomer with fixed stoichiometry (Figure4C, Table1). Thus, mutations in the N-terminal part of HspB6 impair its interaction with HspB1. Both the R/A and ∆ mutations had only a marginal effect on the quaternary structure of HspB5 (Figure5). The heterooligomeric complexes of the WT HspB5 and WT HspB6 were formed only at high temperature and had an apparent molecular weight of 400 kDa and fixed stoichiometry HspB5/HspB6 close to 2:1 (Table1). The R /A mutant of HspB5 also interacted with HspB6 only at elevated temperature and formed heterooligomers with an apparent molecular weight of 350 kDa and stoichiometry HspB5/HspB6 varying in the range of 3:1 to 2:1 (Figure5B). In this case, a larger portion Int. J. Mol. Sci. 2020, 21, 4248 12 of 18 of HspB6 remained free and was not involved in complex formation. Similar results were obtained with the ∆ mutant of HspB5 (Figure5C). Thus, mutations in the N-terminal of HspB5 moderately a ffect its interaction with the WT HspB6. The stoichiometry HspB5/HspB6 in complexes formed by HspB5 mutants was variable and larger than in the case of the WT HspB5, thus leaving larger portions of HspB6 free. The R/A mutant of HspB6 formed heterooligomers with the WT HspB5 only at elevated temperatures (Figure6B). These complexes had a molecular weight of 350 kDa and variable stoichiometry HspB5 /HspB6 (Table1). A rather large portion of HspB6 remained free and was eluted in a separate peak (Figure6B). Similar results were obtained with the ∆ mutant of HspB6. In this case, three poorly separated peaks of heterooligomers were detected in the elution profile (Figure6C). The stoichiometry HspB5 /HspB6 in these complexes varied in the range of 4 to 1 and again a large portion of HspB6 and was not involved in formation of heterooligomeric complexes (Table1). Interestingly,in stark contrast with the exact 1:1 molar ratio of HspB1 and HspB6 in the heterooligomers which can be explained by the preferred formation of heterodimers, the heterooligomers formed by the WT HspB5 and HspB6 reveal a preferred 2:1 molar ratio (Figure5). Several other sHsps were previously shown to heterooligomerize in ratios different from 1:1 (HspB2/HspB3 [37], HspB4/HspB5 [38]) Here, N-terminal mutations in HspB6, but not in HspB5, have a negative effect on the incorporation of HspB6 into the heterooligomers, while the ratio of HspB5 over HspB6 increases up to 3:1 and more (Figure6, Table1). Our attempts to detect the interaction of the WT HspB8 or its N-terminal mutants with the WT HspB1 were unsuccessful (Figure7). Neither at low (data not presented) nor at high temperature did the WT HspB8 or its N-terminal mutants form heterooligomers with the WT HspB1, and each protein was eluted in separate peaks. Similar results were obtained with HspB5 and HspB8 (data not presented). These data agree with our earlier published data obtained by means of chemical crosslinking [25] and disagree with results obtained by means of fluorescence resonance energy transfer (FRET), yeast two hybrid assay or immunoprecipitation [19,20]. The interaction of HspB1 and HspB5 using ion-exchange chromatography (Figure8) revealed heterooligomeric complexes which were formed only at elevated temperatures. The stoichiometry of these complexes was close to 1:1. Neither the R/A nor ∆ mutations of HspB1 or HspB5 had any effect on heterooligomer formation (data not shown). This means that mutations in the N-terminal domain did not affect heterooligomerization of HspB1 and HspB5. The formation of heterooligomeric complexes depends on many factors, such as the oligomeric state of protein partners, the accessibility of sites of interaction and, most importantly, on the presence and quantity of interaction sites. Three sites, namely the α-crystallin domain, C-terminal and N-terminal sites are involved in inter-subunit interaction. The α-Crystallin domain is present in all sHsps, and interaction through this domain is constitutive [39]. HspB1 and HspB5 have conserved IP(I/V) peptide in their C-terminal domain that is involved in inter- and intra-subunit interactions [27,31,40]. This peptide is lacking in the C-terminal domain of HspB6 and HspB8. Finally, it is supposed that the N-terminal domain takes part in formation of heterooligomeric complexes [18,24]. Oversimplifying, we can suppose that HspB1 and HspB5 have three, whereas HspB6 and HspB8 have only two potential sites involved in heterooligomerization. It was suggested that conserved (S/G)RLFD pentapeptide could interact with ACD [41] and by this means drive the interaction of different sHsps. In the case of HspB1 and HspB5, the mutation or deletion of this pentapeptide in either HspB1 or HspB5 will decrease the number of potential binding sites from six (three on HspB1 and three on HspB5) to five. As it was shown in our experiments (Figure8), this small decrease in the number of interaction sites will not dramatically affect the heterooligomerization of these two proteins. In the case of HspB1/HspB6 or HspB5/HspB6, the WT proteins will have five potential sites of interaction (three sites on HspB1 or HspB5 and two sites on HspB6). Mutations in the N-terminal domain of HspB1 or HspB5 will decrease the number of interaction sites from five to four (two sites on HspB1 or HspB5 and two sites on HspB6). In this case, the sites of interaction are equally distributed on sHsps, forming large (HspB1 and HspB5) and small (HspB6) oligomers. Therefore, the WT HspB6 incorporates into large oligomers of HspB1 and HspB5 and forms Int. J. Mol. Sci. 2020, 21, 4248 13 of 18 heterooligomers with definite stoichiometry (Figures3,5 and9). In the pairs formed by the WT HspB1 and HspB5 and mutated HspB6 the number of interaction sites will again be decreased from five to four (three sites on HspB1 and HspB5 and only one site on HspB6). In this case, the subunits of HspB1 and HspB5 tightly interact with each other and HspB6 having only ACD interaction interface will induce partial dissociation of large oligomers formed by HspB1 and HspB5 but will not be able to form heterooligomeric complexes with definite stoichiometry (Figures4,6 and9). In our hands, the interaction of HspB8 with any sHsps is very weak, and therefore we were unable to determine the effect of mutations in the N-terminal domain of this protein on formation of heterooligomers. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 15 of 20

Figure 9. Hypothetical scheme of heterooligomerization of HspB1, HspB5 and HspB6 and their N- Figure 9. Hypothetical scheme of heterooligomerization of HspB1, HspB5 and HspB6 and their N-terminal terminal mutants. HspB1, HspB5 and HspB6 monomers are represented as red, yellow and green half mutants.circles, HspB1, respectively. HspB5 Full and circles HspB6 represent monomers homo- or are heterooligomers represented asand red, the colored yellow area and corresponds green half circles, respectively.to the Fullportion circles of the represent corresponding homo- protein or heterooligomers in heterooligomer. and The the coloredinteraction area of correspondsHspB1 and HspB6. to the portion of the correspondingThe WT HspB1 protein and HspB6 in heterooligomer. form heterooligomeric The interaction complexes of containing HspB1 and similar HspB6. 1:1 Theratio WTof two HspB1 and HspB6 formproteins. heterooligomeric Mutations of HspB1 complexes NTD weakly containing affect similarits interaction 1:1 ratio with of HspB6. two proteins. Mutations Mutations of HspB6 of HspB1 NTD weaklycompromise affect its its interaction interaction with with the WT HspB6. HspB1 Mutations and significant of HspB6 portion compromise of HspB6 was its not interaction included in with the heterooligomers. The interaction of HspB5 and HspB6. The WT HspB5 and HspB6 form definite WT HspB1 and significant portion of HspB6 was not included in heterooligomers. The interaction of heterooligomeric complex with stoichiometry HspB5/HspB6 close to 2:1. Mutations of HspB5 NTD HspB5 and HspB6. The WT HspB5 and HspB6 form definite heterooligomeric complex with stoichiometry weakly affect interaction with HspB6 and the stoichiometry HspB5/HspB6 in heterooligomers was HspB5/largerHspB6 than close 2:1. to Mutations 2:1. Mutations of HspB6 of HspB5compromi NTDse its weakly interaction affect with interaction HspB5 withand different HspB6 and the stoichiometryheterooligomeric HspB5/HspB6 complexes in heterooligomerswith variable stoichiometry was larger were than formed 2:1. Mutations leaving part of HspB6of HspB6 compromise in its interactionhomooligomeric with HspB5 state. and different heterooligomeric complexes with variable stoichiometry were formed leaving part of HspB6 in homooligomeric state. At this moment, little is known about the 3D structure of heterooligomers. The point mutations, and especially the five-residue deletions within the NTD studied here, are likely to affect the contacts made by these domains. In large homooligomers of HspB5 [10] and HspB1 [42], the ACDs associate into dimers, while the NTDs are ordered to a large extent and form trimers. In the case of HspB4 [9] the ACDs still form dimers, whereas the NTDs form either dimeric or tetrameric contacts. With the structure of homooligomers in hand, it is logical to suggest that the NTDs of the two partners would be involved in the direct dimeric, trimeric or tetrameric contacts. However, contacts between the NTD

Int. J. Mol. Sci. 2020, 21, 4248 14 of 18

At this moment, little is known about the 3D structure of heterooligomers. The point mutations, and especially the five-residue deletions within the NTD studied here, are likely to affect the contacts made by these domains. In large homooligomers of HspB5 [10] and HspB1 [42], the ACDs associate into dimers, while the NTDs are ordered to a large extent and form trimers. In the case of HspB4 [9] the ACDs still form dimers, whereas the NTDs form either dimeric or tetrameric contacts. With the structure of homooligomers in hand, it is logical to suggest that the NTDs of the two partners would be involved in the direct dimeric, trimeric or tetrameric contacts. However, contacts between the NTD of one partner and the ACD of another, like those seen in the HspB2/B3 (3:1) heterooligomers [37], are also feasible. Interestingly, the NTD mutations affecting the heterooligomer formation result in changes within this ACD interface (a possibly indirect effect) [24]. The latter observation suggests that the mechanism of mutations located in the NTD on the overall structure and properties of sHsp heterooligomers may be rather complex. Beyond artificial mutations like the ones used here, this observation is also relevant for natural, abundantly observed disease-related mutations [30,43,44].

4. Material and Methods

4.1. Cloning of the Wild-Type Proteins and Their Mutants The cDNA of the WT HspB1, HspB5, HspB6 and HspB8 in pET22b or pET23a [45] were used for obtaining the so-called R/A mutants. Plasmids containing full-size sequences coding R/A mutants HspB1 R27A, HspB5 R22A, HspB6 R27A, HspB8 R29A were obtained in Eurogen. Plasmids containing full size sequences coding the so-called delta mutants of HspB1 ∆26–30, HspB5 ∆21–25, HspB6 ∆26–30 and HspB8 ∆28–32 were also prepared in Eurogen. All constructs were cloned into pET22b(+) or pET23a(+) vectors at NdeI and XhoI sites and integrity and lack of additional mutations were confirmed by DNA sequencing.

4.2. Expression and Purification of Recombinant Proteins The expression of the WT proteins and their mutants was performed in Escherichia coli BL21(DE3) PLysS. For the expression of HspB6 R27A, HspB8 R29A and HspB8 ∆28–32 bacteria were grown on standard Luria-Bertani (LB) media at 37 ◦C. After the optical density at 600 nm reached 0.6–0.8, expression was started by addition of 0.5 mM IPTG and lasted for 6 h. Alternatively, for the production of HspB1 WT, HspB5 WT and its mutants, HspB6 WT, HspB6 ∆26–30 and HspB8 WT bacteria were grown on 3-fold LB media. In this case, bacteria at first were grown for 8 h at 37 ◦C followed by incubation overnight at 30 ◦C. The purification of proteins was performed by earlier described methods [30,35,45]. Briefly, in the case of HspB1 and HspB5, the proteins were subjected to ammonium sulfate fractionation followed by ion-exchange chromatography on HiTrap Q and size-exclusion chromatography on Superdex 200. In the case of HspB6 and HspB8, after ammonium sulfate fractionations, the proteins were subjected to hydrophobic chromatography on HiTrap Phenyl-Sepharose, followed by size-exclusion chromatography on Superdex 200. No less than two preparations of the WT proteins and their mutants were used for this study. The purity of proteins were nor less than 95% according to SDS gel electrophoresis [46] and no significant difference was observed for different batches of the same protein. The yield of recombinant proteins varied between 20 and 120 mg from 1 L of culture. All samples were frozen and stored at 20 C in buffer containing 20 mM Tris-acetate pH 7.6, 10 mM NaCl, 0.1 mM EDTA, 0.1 mM − ◦ phenylmethanesulphonyl fluoride (PMSF) and 2 mM dithiothreitol (DTT). The protein concentration was 0.1% determined spectrophotometrically using A280 equal to 1.775 for HspB1 (UniProtKB P04792), 0.692 for HspB5 (UniProtKB P02489), 0.582 for HspB6 (UniProtKB O14558) and 1.225 for HspB8 (UniProtKB Q9UJY1).

4.3. Formation of Heterooligomeric Complexes

Isolated proteins were reduced by incubation in the presence of 5 mM DTT for 30 min at 37 ◦C. Afterwards, the proteins were pairwise mixed in buffer B (20 mM Tris-acetate, 10 mM NaCl, 150 mM Int. J. Mol. Sci. 2020, 21, 4248 15 of 18

NaCl, 0.1 mM EDTA, 15 mM mercaptoethanol) to obtain equimolar mixture (final concentration of each protein varied in the range of 30–40 µM). Thus, the obtained mixtures were preincubated for 1 h either at 4 ◦C (the rate of subunits exchange is usually negligible low) or at 42 ◦C (the rate of subunit exchange is high). Alternatively, equimolar mixture of analyzed proteins (30–40 µM) after reduction were mixed and incubated in buffer D (20 mM phosphate buffer pH 6.8, 0.1 mM PMSF, 15 mM mercaptoethanol) either at low (4 ◦C) or at high (42 ◦C) temperature for 1 h.

4.4. Analysis of Heterooligomeric Complexes Two methods were used for the detection of the heterooligomeric complexes. In the case of sHsps with different molecular weights, i.e., in the case of HspB1/HspB6, HspB1/HspB8 and HspB5/Hsp6, the formation of heterooligomeric complexes was detected by means of size-exclusion chromatography. Samples (80–100 µL) of isolated proteins or their mixture preincubated either at low or high temperature were loaded on Superdex 200 HR 10/30 column equilibrated with buffer B. The column was run at the rate of 0.5 mL/min and 400 µL samples were collected. The proteins were precipitated by trichloroacetic acid (TCA) and after washing with acetone the protein pellet was dissolved in SDS sample buffer and subjected to SDS gel electrophoresis [46]. After staining and de-staining, the gels were scanned, and intensities of bands were evaluated by using Gel Analyzer program. The column was calibrated by using the following protein standards: thyroglobulin (669 kDa), ferritin (440 kDa), glyceraldehyde-3-phosphate dehydrogenase (144 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa) and RNAse (13.7 kDa). In the case of sHsps with similar large molecular weights, i.e., in the case of HspB1/HspB5, the formation of heterooligomeric complexes was determined by means of ion-exchange chromatography. Theoretically determined isoelectric points of HspB1 and HspB5 are equal to 5.98 and 6.76, respectively. Therefore, at pH 6.8, HspB1 will be tightly bound, whereas HspB5 will not be bound to the DEAE HiTrap column. Under these conditions, the heterooligomeric complexes formed by these proteins will have intermediate charge and will be weakly bound on the DEAE HiTrap column. Isolated proteins or their mixture (80–100 µL) were loaded on 1 mL DEAE HiTrap column equilibrated with buffer D (20 mM phosphate buffer pH 6.8, 0.1 mM PMSF, 15 mM mercaptoethanol). The column was washed with 5 column volumes of buffer D and eluted with 20 column volumes of linear salt gradient (0–500 mM NaCl). The column was run at the rate of 1 mL/min and 400 µL fractions were collected. Proteins were precipitated with TCA, protein pellet was washed with acetone, dissolved in SDS sample buffer and subjected to SDS gel electrophoresis. After staining and de-staining, the gels were scanned, and the intensity of each protein band was determined by the Gel Analyzer program. The effectiveness of heterooligomerization was analyzed using a correlation coefficient: P AiBi Corr = p p (1) P 2 P 2 Ai Bi where Ai and Bi are intensities of protein bands in fraction i.

Author Contributions: V.M.S. and N.B.G. conducted the experiments, V.M.S. prepared all figures, N.B.G. and S.V.S. prepared and edited the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This investigation was supported by the Russian Foundation for Basic Research (grant 19-04-00038 to N.B.G.) and by the Research Foundation, Flanders (grants G093615N and WO03315N to S.V.S.) Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Int. J. Mol. Sci. 2020, 21, 4248 16 of 18

Abbreviations

DTT dithiothreitol ME mercaptoethanol PMSF phenylmethanesulphonyl fluoride SDS sodium dodecyl sulfate SEC size-exclusion chromatography TCA trichloroacetic acid WT wild type

References

1. Haslbeck, M.; Vierling, E. A first line of stress defense: Small heat shock proteins and their function in protein homeostasis. J. Mol. Biol. 2015, 427, 1537–1548. [CrossRef][PubMed] 2. Janowska, M.K.; Baughman, H.E.R.; Woods, C.N.; Klevit, R.E. Mechanisms of small heat shock proteins. Cold Spring Harb. Perspect. Biol. 2019, 11, a034025. [CrossRef][PubMed] 3. Bakthisaran, R.; Tangirala, R.; Rao, C.M. Small heat shock proteins: Role in cellular functions and pathology. Biochim. Biophys. Acta 2015, 1854, 291–319. [CrossRef] 4. Fontaine, J.M.; Rest, J.S.; Welsh, M.J.; Benndorf, R. The sperm outer dense fiber protein is the 10th member of the superfamily of mammalian small stress proteins. Cell Stress Chaperones 2003, 8, 62–69. [CrossRef] 5. Kappe, G.; Franck, E.; Verschuure, P.; Boelens, W.C.; Leunissen, J.A.; de Jong, W.W. The encodes 10 alpha-crystallin-related small heat shock proteins: Hspb1-10. Cell Stress Chaperones 2003, 8, 53–61. [CrossRef] 6. Vos, M.J.; Kanon, B.; Kampinga, H.H. Hspb7 is a sc35 speckle resident small . Biochim. Biophys. Acta 2009, 1793, 1343–1353. [CrossRef][PubMed] 7. Taylor, R.P.; Benjamin, I.J. Small heat shock proteins: A new classification scheme in mammals. J. Mol. Cell. Cardiol. 2005, 38, 433–444. [CrossRef][PubMed] 8. Fang, X.; Bogomolovas, J.; Trexler, C.; Chen, J. The bag3-dependent and -independent roles of cardiac small heat shock proteins. JCI Insight 2019, 4, e126464. [CrossRef] 9. Kaiser, C.J.O.; Peters, C.; Schmid, P.W.N.; Stavropoulou, M.; Zou, J.; Dahiya, V.; Mymrikov, E.V.; Rockel, B.; Asami, S.; Haslbeck, M.; et al. The structure and oxidation of the eye lens chaperone alphaa-crystallin. Nat. Struct. Mol. Biol. 2019, 26, 1141–1150. [CrossRef] 10. Braun, N.; Zacharias, M.; Peschek, J.; Kastenmuller, A.; Zou, J.; Hanzlik, M.; Haslbeck, M.; Rappsilber, J.; Buchner, J.; Weinkauf, S. Multiple molecular architectures of the eye lens chaperone alphab-crystallin elucidated by a triple hybrid approach. Proc. Natl. Acad. Sci. USA 2011, 108, 20491–20496. [CrossRef] 11. Kriehuber, T.; Rattei, T.; Weinmaier, T.; Bepperling, A.; Haslbeck, M.; Buchner, J. Independent evolution of the core domain and its flanking sequences in small heat shock proteins. FASEB J. 2010, 24, 3633–3642. [CrossRef][PubMed] 12. Suzuki, A.; Sugiyama, Y.; Hayashi, Y.; Nyu-i, N.; Yoshida, M.; Nonaka, I.; Ishiura, S.; Arahata, K.; Ohno, S. Mkbp, a novel member of the small heat shock protein family, binds and activates the myotonic dystrophy protein kinase. J. Cell Biol. 1998, 140, 1113–1124. [CrossRef][PubMed] 13. Sugiyama, Y.; Suzuki, A.; Kishikawa, M.; Akutsu, R.; Hirose, T.; Waye, M.M.; Tsui, S.K.; Yoshida, S.; Ohno, S. Muscle develops a specific form of small heat shock protein complex composed of mkbp/ and hspb3 during myogenic differentiation. J. Biol. Chem. 2000, 275, 1095–1104. [CrossRef][PubMed] 14. Mymrikov, E.V.; Riedl, M.; Peters, C.; Weinkauf, S.; Haslbeck, M.; Buchner, J. Regulation of small heat-shock proteins by hetero-oligomer formation. J. Biol. Chem. 2020, 295, 158–169. [CrossRef] 15. Zantema, A.; Verlaan-De Vries, M.; Maasdam, D.; Bol, S.; van der Eb, A. Heat shock protein 27 and alpha b-crystallin can form a complex, which dissociates by heat shock. J. Biol. Chem. 1992, 267, 12936–12941. 16. Kato, K.; Shinohara, H.; Goto, S.; Inaguma, Y.; Morishita, R.; Asano, T. Copurification of small heat shock protein with alpha b crystallin from human skeletal muscle. J. Biol. Chem. 1992, 267, 7718–7725. [PubMed] 17. Kato, K.; Goto, S.; Inaguma, Y.; Hasegawa, K.; Morishita, R.; Asano, T. Purification and characterization of a 20-kda protein that is highly homologous to alpha b crystallin. J. Biol. Chem. 1994, 269, 15302–15309. 18. Delbecq, S.P.; Rosenbaum, J.C.; Klevit, R.E. A mechanism of subunit recruitment in human small heat shock protein oligomers. Biochemistry 2015, 54, 4276–4284. [CrossRef] Int. J. Mol. Sci. 2020, 21, 4248 17 of 18

19. Fontaine, J.M.; Sun, X.; Benndorf, R.; Welsh, M.J. Interactions of hsp22 () with hsp20, alphab-crystallin, and hspb3. Biochem. Biophys. Res. Commun. 2005, 337, 1006–1011. [CrossRef] 20. Sun, X.; Fontaine, J.M.; Rest, J.S.; Shelden, E.A.; Welsh, M.J.; Benndorf, R. Interaction of human hsp22 (hspb8) with other small heat shock proteins. J. Biol. Chem. 2004, 279, 2394–2402. [CrossRef] 21. Bukach, O.V.; Seit-Nebi, A.S.; Marston, S.B.; Gusev, N.B. Some properties of human small heat shock protein hsp20 (hspb6). Eur. J. Biochem. 2004, 271, 291–302. [CrossRef][PubMed] 22. Bukach, O.V.; Glukhova, A.E.; Seit-Nebi, A.S.; Gusev, N.B. Heterooligomeric complexes formed by human small heat shock proteins hspb1 () and hspb6 (hsp20). Biochim. Biophys. Acta 2009, 1794, 486–495. [CrossRef][PubMed] 23. Heirbaut, M.; Lermyte, F.; Martin, E.M.; Beelen, S.; Verschueren, T.; Sobott, F.; Strelkov, S.V.; Weeks, S.D. The preferential heterodimerization of human small heat shock proteins hspb1 and hspb6 is dictated by the n-terminal domain. Arch. Biochem. Biophys. 2016, 610, 41–50. [CrossRef] 24. Heirbaut, M.; Lermyte, F.; Martin, E.M.; Beelen, S.; Sobott, F.; Strelkov, S.V.; Weeks, S.D. Specific sequences in the n-terminal domain of human small heat-shock protein hspb6 dictate preferential hetero-oligomerization with the orthologue hspb1. J. Biol. Chem. 2017, 292, 9944–9957. [CrossRef] 25. Mymrikov, E.V.; Seit-Nebi, A.S.; Gusev, N.B. Heterooligomeric complexes of human small heat shock proteins. Cell Stress Chaperones 2012, 17, 157–169. [CrossRef] 26. Arrigo, A.P. Human small heat shock proteins: Protein interactomes of homo- and hetero-oligomeric complexes: An update. FEBS Lett. 2013, 587, 1959–1969. [CrossRef][PubMed] 27. Hochberg, G.K.; Benesch, J.L. Dynamical structure of alphab-crystallin. Prog. Biophys. Mol. Biol. 2014, 115, 11–20. [CrossRef][PubMed] 28. Hilton, G.R.; Lioe, H.; Stengel, F.; Baldwin, A.J.; Benesch, J.L. Small heat-shock proteins: Paramedics of the cell. Top. Curr. Chem. 2012, 328, 69–98. 29. Jehle, S.; Vollmar, B.S.; Bardiaux, B.; Dove, K.K.; Rajagopal, P.; Gonen, T.; Oschkinat, H.; Klevit, R.E. N-terminal domain of alphab-crystallin provides a conformational switch for multimerization and structural heterogeneity. Proc. Natl. Acad. Sci. USA 2011, 108, 6409–6414. [CrossRef] 30. Muranova, L.K.; Weeks, S.D.; Strelkov, S.V.; Gusev, N.B. Characterization of mutants of human small heat shock protein hspb1 carrying replacements in the n-terminal domain and associated with hereditary motor neuron diseases. PLoS ONE 2015, 10, e0126248. [CrossRef] 31. Clouser, A.F.; Baughman, H.E.; Basanta, B.; Guttman, M.; Nath, A.; Klevit, R.E. Interplay of disordered and ordered regions of a human small heat shock protein yields an ensemble of ‘quasi-ordered’ states. eLife 2019, 8, eLife50259. [CrossRef][PubMed] 32. Peschek, J.; Braun, N.; Rohrberg, J.; Back, K.C.; Kriehuber, T.; Kastenmuller, A.; Weinkauf, S.; Buchner, J. Regulated structural transitions unleash the chaperone activity of alphab-crystallin. Proc. Natl. Acad. Sci. USA 2013, 110, E3780–E3789. [CrossRef][PubMed] 33. Heirbaut, M.; Beelen, S.; Strelkov, S.V.; Weeks, S.D. Dissecting the functional role of the n-terminal domain of the human small heat shock protein hspb6. PLoS ONE 2014, 9, e105892. [CrossRef][PubMed] 34. Weeks, S.D.; Muranova, L.K.; Heirbaut, M.; Beelen, S.; Strelkov, S.V.; Gusev, N.B. Characterization of human small heat shock protein hspb1 alpha-crystallin domain localized mutants associated with hereditary motor neuron diseases. Sci. Rep. 2018, 8, 1–15. [CrossRef][PubMed] 35. Shatov, V.M.; Weeks, S.D.; Strelkov, S.V.; Gusev, N.B. The role of the arginine in the conserved n-terminal domain rlfdqxfg motif of human small heat shock proteins hspb1, hspb4, hspb5, hspb6, and hspb8. Int. J. Mol. Sci. 2018, 19, 2112. [CrossRef] 36. Rogalla, T.; Ehrnsperger, M.; Preville, X.; Kotlyarov, A.; Lutsch, G.; Ducasse, C.; Paul, C.; Wieske, M.; Arrigo, A.P.; Buchner, J.; et al. Regulation of hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor alpha by phosphorylation. J. Biol. Chem. 1999, 274, 18947–18956. [CrossRef] 37. Clark, A.R.; Vree Egberts, W.; Kondrat, F.D.L.; Hilton, G.R.; Ray, N.J.; Cole, A.R.; Carver, J.A.; Benesch, J.L.P.; Keep, N.H.; Boelens, W.C.; et al. Terminal regions confer plasticity to the tetrameric assembly of human hspb2 and hspb3. J. Mol. Biol. 2018, 430, 3297–3310. [CrossRef][PubMed] 38. Horwitz, J. Alpha-crystallin. Exp. Eye Res. 2003, 76, 145–153. [CrossRef] 39. Clark, A.R.; Lubsen, N.H.; Slingsby, C. Shsp in the eye lens: Crystallin mutations, cataract and proteostasis. Int. J. Biochem. Cell Biol. 2012, 44, 1687–1697. [CrossRef][PubMed] Int. J. Mol. Sci. 2020, 21, 4248 18 of 18

40. Delbecq, S.P.; Jehle, S.; Klevit, R. Binding determinants of the small heat shock protein, alphab-crystallin: Recognition of the ‘ixi’ motif. EMBO J. 2012, 31, 4587–4594. [CrossRef][PubMed] 41. Klevit, R.E. Peeking from behind the veil of enigma: Emerging insights on small heat shock protein structure and function. Cell Stress Chaperones 2020.[CrossRef] 42. Nappi, L.; Aguda, A.H.; Nakouzi, N.A.; Lelj-Garolla, B.; Beraldi, E.; Lallous, N.; Thi, M.; Moore, S.; Fazli, L.; Battsogt, D.; et al. Ivermectin inhibits hsp27 and potentiates efficacy of oncogene targeting in tumor models. J. Clin. Investig. 2020, 130, 699–714. [CrossRef][PubMed] 43. Datskevich, P.N.; Nefedova, V.V.; Sudnitsyna, M.V.; Gusev, N.B. Mutations of small heat shock proteins and human congenital diseases. Biochemistry 2012, 77, 1500–1514. [CrossRef][PubMed] 44. Muranova, L.K.; Ryzhavskaya, A.S.; Sudnitsyna, M.V.; Shatov, V.M.; Gusev, N.B. Small heat shock proteins and human neurodegenerative diseases. Biochemistry 2019, 84, 1256–1267. [CrossRef][PubMed] 45. Mymrikov, E.V.; Bukach, O.V.; Seit-Nebi, A.S.; Gusev, N.B. The pivotal role of the beta 7 strand in the intersubunit contacts of different human small heat shock proteins. Cell Stress Chaperones 2010, 15, 365–377. [CrossRef][PubMed] 46. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage t4. Nature 1970, 227, 680–685. [CrossRef][PubMed]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).