Evaluating the Effect of Phosphorylation on the Structure and Dynamics of Hsp27 Dimers by Means of Ion Mobility Mass Spectrometry

The quaternary structure and dynamics of the human small heat-shock protein Hsp27 are linked to its molecular chaperone function and influenced by post-translational modifications, including phosphorylation. Phosphorylation of Hsp27 promotes oligomer dissociation and can enhance chaperone activity. This study explored the impact of phosphorylation on the quaternary structure and dynamics of Hsp27. Using mutations that mimic phosphorylation, and ion mobility mass spectrometry, we show that successive substitutions result in an increase in the conformational heterogeneity of Hsp27 dimers. In contrast, we did not detect any changes in the structure of an Hsp27 12-mer, representative of larger Hsp27 oligomers. Our data suggest that oligomer dissociation and increased flexibility of the dimer contribute to the enhanced chaperone activity of phosphorylated Hsp27. Thus, post-translational modifications such as phosphorylation play a crucial role in modulating both the tertiary and quaternary structure of Hsp27, which is pivotal to its function as a key component of the proteostasis network in cells. Our data demonstrate the utility of ion mobility mass spectrometry for probing the structure and dynamics of heterogeneous proteins

Biophysical characterization of wild-type and mutant SEN.

<p>(<b>A</b>) Far-UV circular dichroism spectra demonstrating the predominantly α-helical secondary structure of recombinant wild-type SEN (solid), SEN^K312A (dashed) and SEN^K362A (dotted). (<b>B</b>) Comparison of the thermal stability of wild-type SEN (black), SEN^K312A (striped) and SEN^K362A (clear) determined by the thermofluor assay. The assay was performed in triplicate and error bars indicate the standard deviation.</p

Size-exclusion chromatography and native mass spectrometry analysis of SEN^K362A.

<p>(<b>A</b>) Size-exclusion chromatography elution profile showing that the protein elutes as two peaks. Molecular weight standards are indicated by triangles. (<b>B</b>) Mass spectrum illustrating that elution peak 1 is composed of a mixture of octameric, heptameric (*) and monomeric (o) species. (<b>C</b>) Mass spectrum showing that elution peak 2 contains monomeric species only.</p

K362 interactions in wild-type SEN.

<p>(<b>A</b>) SEN minor interface with residues interacting with K362 shown as stick representations. (<b>B</b>) Close-up view of the area bounded by a dashed line in <i>(A)</i> showing the potential hydrogen bond network involving K362, T389 and N390 of the same monomer, and E359’ and E363’ of the opposing monomer.</p

Size-exclusion chromatography and mass spectrometry analysis of wild-type SEN and SEN^K312A.

<p>Size-exclusion chromatography elution profile of (<b>A</b>) wild-type SEN and (<b>B</b>) SEN^K312A shows that both proteins elute as a single mono-disperse peak. Molecular weight standards are indicated by triangles. Subsequent mass spectra of (<b>C</b>) wild-type SEN and (<b>D</b>) SEN^K312A illustrate that both proteins exist as octamers.</p

Enzymatic activity of recombinant wild-type SEN and mutant forms.

<p>The graph shown was constructed from the averages of three independent experiments, with error bars indicating the standard deviation: wild-type SEN (solid), SEN^K312A (dashed) and SEN^K362A(dotted).</p

Small-angle X-ray scattering (SAXS) data.

<p>(<b>A</b>) Pairwise interatomic distance distribution derived from SAXS. The maximum distance (<i>D</i>_max) from the <i>P</i>(<i>r</i>) is 157 Å, and the radius of gyration (<i>R</i>_g) is 50.6 Å. (<b>B</b>) Experimental scattering of wild-type SEN on absolute, logarithmic scale, with 1<i>σ</i> error bars shown in gray. Theoretical fits of the <i>ab initio</i> models (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121764#pone.0121764.g001" target="_blank">Fig. 1C</a>) are shown in blue for the dummy residue model, and purple for the cluster-representative bead model. (<b>C</b>) Guinier transformation of the data from a concentration series, demonstrating concentration-dependent <i>R</i>_g variation of &lt; 5% and linearity over the region <i>q</i>.<i>R</i>_g &lt; 1.3.</p

Crystallographic data collection and refinement statistics.

<p>^<i>a</i> Each dataset was collected from a single crystal; the values in parentheses are for the highest-resolution shell. <i>R</i>_meas = <i>∑</i>_<i>hkl</i>(<i>N</i>(<i>hkl</i>)/[<i>N</i>(<i>hkl</i>)-1])^1/2 ∑_<i>i</i>|I_<i>i</i>(<i>hkl</i>)- &lt;<i>I</i>(<i>hkl</i>)&gt;|/ ∑_<i>hkl</i>∑_<i>i</i>I_<i>i</i>(<i>hkl</i>), where <i>I</i>_<i>i</i>(<i>hkl</i>) is the intensity of the <i>i</i>th measurement of an equivalent reflection with indices <i>hkl</i>.</p><p>Crystallographic data collection and refinement statistics.</p

Forward primers used for PCR construction and DNA sequence analysis of SEN site-directed mutants.

<p>Forward primers used for PCR construction and DNA sequence analysis of SEN site-directed mutants.</p

Molecular docking results and mechanism of plasminogen binding by SEN.

<p>(<b>A</b>) Human plasminogen KR1 docked at the SEN minor interface. Plasminogen domains are colored as follows: pan-apple (PAp) domain blue; KR1 red; KR2 yellow; KR3 orange; KR4 green; KR5 purple; serine protease (SP) domain cyan. Linear three-residue patches on KR1 and KR5 implicated in lysine binding are shown as spheres. One SEN dimer is colored green and pink, respectively, with the remaining octamer colored in shades of grey. One occurrence of SEN BS1 and BS2 is shown as blue and red spheres, respectively. (<b>B</b>) Proposed model for the binding of plasminogen by SEN involving interactions between BS1 and KR1, and BS2 and KR5. The color scheme follows <i>(A)</i>. Figure adapted from Law <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121764#pone.0121764.ref022" target="_blank">22</a>].</p