The effect of the N-terminal HEAT repeats of the A-subunit on methylation.

<p>(<b>A</b>) Schematic representation of internal truncations of the N-terminal HEAT repeats to reduce the length of the A-subunit N-terminal structure. (<b>B</b>) Normalized concentrations of PP2Ac assembled with full-length or truncated A-subunits used for kinetic analysis of PP2A methylation in (<b>C</b>). (<b>C</b>) Kinetics of methylation of PP2A core enzymes with varied length of the A-subunit. Results from 6–7 independent experiments were summarized and the average K_m ± std. dev. was calculated and shown in the table on the right. (<b>D</b>) Correlation between the number of deleted N-terminal HEAT repeats and the K_m of methylation of PP2A core enzyme by LCMT-1. Statistical test of this correlation was performed and the P value was calculated and shown. (<b>E</b>) Structure of the core enzyme portion of PP2A holoenzyme (PDB ID: 2NPP). The A-subunit, PP2Ac, and PP2Ac-tail are colored green, blue, and magenta, respectively. The positions of the HEAT-repeats where truncations were made are indicated by numbers.</p

Electrostatic interactions between the A-subunit and LCMT-1.

<p>(<b>A</b>) Structural model of the PP2A core enzyme-LCMT-1 complex built from the structure of the PP2Ac-LCMT-1 complex and the A-subunit with morphed conformation. The electrostatic potential for the A-subunit (left panel) and LCMT-1 (right panel) is shown. The corresponding regions on the A-subunit and LCMT-1 for potential electrostatic interactions are circled (i and ii). The A-subunit residues that directly contact LCMT-1 in the model, Arg183 and Arg258, are shown. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086955#pone.0086955.s003" target="_blank">Movie S1</a>. (<b>B</b>) The effect of the A-subunit mutations to Arg183 and Arg258 on methylation of the PP2A core enzyme. Results from 5–7 independent experiments were summarized and the average K_m ± std. dev. and the P value were calculated and shown. <b>(C)</b> FRET assay measured changes in the distance between the N- and C-termini of the A-subunit in the core enzyme prior to and after addition of an excess molar amount of LCMT-1 or a stoichiometric amount of PR70 (108–575). Representative results were shown with mean ± SEM calculated from triplicate.</p

Mechanisms of the Scaffold Subunit in Facilitating Protein Phosphatase 2A Methylation

<div><p>The function of the biologically essential protein phosphatase 2A (PP2A) relies on formation of diverse heterotrimeric holoenzymes, which involves stable association between PP2A scaffold (A) and catalytic (C or PP2Ac) subunits and binding of variable regulatory subunits. Holoenzyme assembly is highly regulated by carboxyl methylation of PP2Ac-tail; methylation of PP2Ac and association of the A and C subunits are coupled to activation of PP2Ac. Here we showed that PP2A-specific methyltransferase, LCMT-1, exhibits a higher activity toward the core enzyme (A–C heterodimer) than free PP2Ac, and the A-subunit facilitates PP2A methylation via three distinct mechanisms: 1) stabilization of a proper protein fold and an active conformation of PP2Ac; 2) limiting the space of PP2Ac-tail movement for enhanced entry into the LCMT-1 active site; and 3) weak electrostatic interactions between LCMT-1 and the N-terminal HEAT repeats of the A-subunit. Our results revealed a new function and novel mechanisms of the A-subunit in PP2A methylation, and coherent control of PP2A activity, methylation, and holoenzyme assembly.</p></div

The A-subunit enhances PP2A methylation and interaction with LCMT-1.

<p>(<b>A</b>) Influence of the A-subunit, mini-A, and PTPA on PP2A methylation, tested by addition of these proteins in a stoichiometric amount to PP2Ac, prior to determination of the kinetics of PP2A methylation. Representative results were shown with mean ± SEM calculated from triplicate (upper figure panel). Statistical analysis of 4–12 independent experiments was performed and the average K_m ± std. dev. and the P value were calculated and summarized in the table below. (<b>B</b>) Binding affinity between LCMT-1, preloaded with SAH, and unmethylated, methylated PP2A core enzyme or free PP2Ac, measured by ITC.</p

Shortening of PP2Ac-tail enhanced methylation.

<p>(<b>A</b>) Schematic representation of Δ294–298 truncation of PP2Ac and the range of PP2Ac-tail mobility. PP2Ac is shown as blue sphere with two small red spheres representing catalytic metal ions. The PP2Ac-tail is shown in cyan. The cyan spheres surrounding PP2Ac indicate the range of movement of PP2Ac-tail. Note that the Δ294–298 truncation reduces the space for the PP2Ac-tail movement. (<b>B</b>) Kinetics of methylation of PP2Ac Δ294–298 and full-length PP2Ac either as the free catalytic subunit or as the core enzymes assembled with the A-subunit with internal deletion of increasing number of N-terminal HEAT repeats. Results from 3–9 independent experiments were summarized and the average K_m ± std. dev. and the P value were calculated and shown. (<b>C</b>) Kinetics of methylation of PP2Ac Δ294–298 associated with the A-subunit with internal deletion of increasing number of N-terminal HEAT repeats (left) and comparison of the full-length PP2Ac and PP2Ac Δ294–298 in correlation of the K_m of methylation and the number of N-terminal HEAT repeats of the A-subunit deleted in the core enzymes (right). Statistical test of this correlation was performed and P values were calculated and shown.</p

Structural Insights into the Tumor-Promoting Function of the MTDH-SND1 Complex

Metadherin (MTDH) and Staphylococcal nuclease domain containing 1 (SND1) are overexpressed and interact in diverse cancer types. The structural mechanism of their interaction remains unclear. Here, we determined the high-resolution crystal structure of MTDH-SND1 complex, which reveals an 11-residue MTDH peptide motif occupying an extended protein groove between two SN domains (SN1/2), with two MTDH tryptophan residues nestled into two well-defined pockets in SND1. At the opposite side of the MTDH-SND1 binding interface, SND1 possesses long protruding arms and deep surface valleys that are prone to binding with other partners. Despite the simple binding mode, interactions at both tryptophan-binding pockets are important for MTDH and SND1’s roles in breast cancer and for SND1 stability under stress. Our study reveals a unique mode of interaction with SN domains that dictates cancer-promoting activity and provides a structural basis for mechanistic understanding of MTDH-SND1-mediated signaling and for exploring therapeutic targeting of this complex