Structural Basis for the Interaction of a Human Small Heat Shock Protein with the 14-3-3 Universal Signaling Regulator

Abstract

Summary By interacting with hundreds of protein partners, 14-3-3 proteins coordinate vital cellular processes. Phosphorylation of the small heat shock protein HSPB6 within its intrinsically disordered N-terminal domain activates its interaction with 14-3-3, ultimately triggering smooth muscle relaxation. After analyzing the binding of an HSPB6-derived phosphopeptide to 14-3-3 using isothermal calorimetry and X-ray crystallography, we have determined the crystal structure of the complete assembly consisting of the 14-3-3 dimer and full-length HSPB6 dimer and further characterized this complex in solution using fluorescence spectroscopy, small-angle X-ray scattering and limited proteolysis. We show that selected intrinsically disordered regions of HSPB6 are transformed into well-defined conformations upon the interaction, whereby an unexpectedly asymmetric structure is formed. This structure provides the first-ever atomic resolution snapshot of a human small HSP in functional state, explains how 14-3-3 proteins sequester their regulatory partners, and can inform the design of small-molecule interaction modifiers to be used as myorelaxants.

Molecular basis of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1

“…Interestingly, the salt bridge between pNth1 1-751 residues R81 and D85 is responsible for an abrupt change in the direction of the polypeptide chain (Figs. 2B and 3B), thus again mimicking the role of the Pro residue at the +2 position (9,25,27,28). The intervening linker sequence between the two 14-3-3 binding motifs of pNth1 (residues 64−79) is ordered compared with known structures of 14-3-3 protein complexes, most likely a result of numerous interactions with helices A1 and A3 of Bmh1 (Fig.…”

“…3). Interactions between pNth1 1-751 residues D63 and N64 at the +3 and +4 positions and Bmh1 residues K51 and N52 appear to force a change in the direction of the polypeptide chain, mimicking the role of the Pro residue, which is frequently found in 14-3-3 binding motifs at the +2 position relative to the phosphorylated residue (9,(25)(26)(27)(28). The second 14-3-3 binding motif of pNth1 (sequence RRGpS 83 ED) also does not contain a Pro residue at the +2 position relative to the phosphorylated residue (Fig.…”

“…Comparison of the Bmh1:pNth1 1-751 , 14-3-3ζ:phosphopeptide, 14-3-3ζ:AANAT, and 14-3-3σ:heat shock protein beta-6 complexes. (A) Comparison of the main-chain conformation of the first 14-3-3 binding motif of pNth1 (sequence RTRTMpS 60 VFDN, shown in cyan) with a "mode 2" 14-3-3 peptide (sequence RLYHpSLPA, PDB ID 1QJA, shown in green) (25) and the 14-3-3 binding motifs of AANAT (sequence QRRHpTLPA, PDB ID 1IB1, shown in orange) (9) and heat shock protein beta-6 (sequence LRRApSAPL, PDB ID 5LTW, shown in red) (27). The C-terminal portion of the 14-3-3 binding motifs is indicated by black ellipse.…”

“…2B). As a result of these interactions, the C-terminal portions of both pNth1 motifs leave the binding grooves in similar orientation as polypeptide chains of other proteins and peptides including, for example, AANAT (9), the heat shock protein beta-6 (27), and short phosphopeptides (25,28), that were cocrystalized with 14-3-3 and whose 14-3-3 binding motifs contain proline residue at the +2 position (Fig. 3).…”

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

“…There is evidence from interactome studies that the Nterminal region of plant sHsps is involved in interacting with amorphously aggregating target proteins (Jaya et al 2009). Another example of the unstructured Nterminal region of sHsps' involvement in interacting with other proteins comes from the recent work of Sluchanko et al (2017) who determined the crystal structure of a complex between a phosphorylated form (at Ser16) of the dimeric sHsp, Hsp20 (van de Klundert et al 1998;Weeks et al 2014) and the 14-3-3σ dimer, i.e. the two proteins form a 2:2 complex.…”