International Journal of Molecular Sciences Article The Heterooligomerization of Human Small Heat Shock Proteins 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 (D 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 D 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 D 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 genes 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 (D 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 andtheirD 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 andD 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 D 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