αB-Crystallin: A Hybrid Solid-State/Solution-State NMR Investigation Reveals Structural Aspects of the Heterogeneous Oligomer

Abstract

SummaryAtomic level structural information on αB-Crystallin (αB), a prominent member of the small Heat Shock Protein (sHSP) family has been a challenge to obtain due its polydisperse, oligomeric nature. We show that magic-angle spinning solid-state NMR can be used to obtain high-resolution information on ∼ 580 kDa human αB assembled from 175-residue, 20 kDa subunits. An ∼100-residue α-crystallin domain is common to all sHSPs and solution-state NMR was performed on two different α-crystallin domain constructs isolated from αB. In vitro, the chaperone-like activities of full-length αB and the isolated α-crystallin domain are identical. Chemical shifts of the backbone and the C β resonances have been obtained for residues 64-162 (α-crystallin domain plus part of the C-terminus) in αB and the isolated α-crystallin domain by solid-and solution-state NMR, respectively. Both sets of data strongly predict six β-strands in the α-crystallin domain. A majority of residues in the α-crystallin domain have similar chemical shifts in both solid-and solution-state indicating a similar structure for the domain in its isolated and oligomeric forms. Sites of inter-subunit interaction are identified from chemical shift differences that cluster to specific regions of the α-crystallin domain. Multiple signals are observed for the resonances of M68 in the oligomer, identifying the region containing this residue as existing in heterogeneous environments within αB. Evidence for a novel dimerization motif in the human α-crystallin domain is obtained by a comparison of (i) solid-and solution-state chemical shift data and (ii) 1 H-15 N HSQC spectra as a function of pH. The isolated α-crystallin domain undergoes a dimer-monomer transition over the pH range of 7.5 to 6.8. This steep pH-dependent switch may be important for αB to function optimally, e.g., to preserve the filament integrity of cardiac muscle proteins such as actin and desmin during cardiac ischemia which is accompanied by acidosis.

Crystal structures of truncated alphaA and alphaB crystallins reveal structural mechanisms of polydispersity important for eye lens function

“…This antiparallel betasheet interaction was suggested previously by sitedirected labeling EPR studies and by hybrid solidstate/solution-state NMR. [27][28][29][30] The structures reported here extend the information provided by the structures of Bagneris et al 26 They offer electron density for the important residues 151-157, which the ABC 67-157 structure lacks, and also offer the structure of the highly conserved C-terminal tail sequence IPI/V, which illuminates one factor in maintenance of lens transparency. Related structures of other members of the small heat shock protein (sHSP) family [15][16][17] have been determined: for Wheat Hsp16.9 (WhHsp16.9), 31 Methanococcus janaschii Hsp16.5 (MjHsp16.5), 32 Xanthomonas axonopodis HspA, 33 and Metazoan Tsp36.…”

The pivotal role of the β7 strand in the intersubunit contacts of different human small heat shock proteins

“…Intersubunit contacts were analyzed either by indirect methods or using different fragments of sHsp and predominantly for αB-crystallin and to a smaller extent for HspB1 (Berengian et al 1999;Ghosh and Clark 2005;Jehle et al 2009;Feil et al 2001), whereas all other human sHsp remain poorly investigated. We tried to analyze the intersubunit contacts of three human sHsp by inserting the single Cys residue in a position homologous to that of Cys137 of human HspB1.…”

“…To overcome this problem, cryo-electron microscopy (Haley et al 2000), site-directed spin labeling (Mchaourab et al 2009;Berengian et al 1999), and protein pin array technique (Ghosh and Clark, 2005) were used for investigation of the structure of full-size human sHsp. In addition, the structure of isolated α-crystallin domain of human sHsp was analyzed by means of hybrid solid-solution NMR spectroscopy (Jehle et al 2009), synchrotron radiation Xray scattering (Feil et al 2001), and X-ray crystallography (Bagneris et al 2009). …”

“…Moreover, there are large differences even in the structure of isolated α-crystallin domains determined by different methods (Feil et al 2001;Jehle et al 2009;Bagneris et al 2009) and significant differences in the detail of dimer structure of crystallin domains derived from different sHsp (Bagneris et al 2009). Finally, the data obtained up to now predominantly illuminate structural properties of human αB-crystallin (HspB5) and to a smaller extent of HspB1 and HspB6.…”

The functional roles of the unstructured N- and C-terminal regions in αB-crystallin and other mammalian small heat-shock proteins

“…X-ray crystallographic studies have determined the atomic-level structure of the excised α-crystallin domain (ACD) of sHsps which encompasses approximately the central 80 amino acids of the protein. Solid state NMR studies have also provided such information about the ACD, in addition to some detail about the structural arrangement of the N-terminal region (Jehle et al 2009;Jehle et al 2011). However, the atomic-level structure of the N-and C-terminal regions and their relationship to the Electronic supplementary material The online version of this article (doi:10.1007/s12192-017-0789-6) contains supplementary material, which is available to authorized users.…”