Examining post‐translational modification‐mediated protein–protein interactions using a chemical proteomics approach

Post‐translational modifications (PTM) of proteins can control complex and dynamic cellular processes via regulating interactions between key proteins. To understand these regulatory mechanisms, it is critical that we can profile the PTM‐dependent protein–protein interactions. However, identifying these interactions can be very difficult using available approaches, as PTMs can be dynamic and often mediate relatively weak protein–protein interactions. We have recently developed CLASPI (cross‐linking‐assisted and stable isotope labeling in cell culture‐based protein identification), a chemical proteomics approach to examine protein–protein interactions mediated by methylation in human cell lysates. Here, we report three extensions of the CLASPI approach. First, we show that CLASPI can be used to analyze methylation‐dependent protein–protein interactions in lysates of fission yeast, a genetically tractable model organism. For these studies, we examined trimethylated histone H3 lysine‐9 (H3K9Me₃)‐dependent protein–protein interactions. Second, we demonstrate that CLASPI can be used to examine phosphorylation‐dependent protein–protein interactions. In particular, we profile proteins recognizing phosphorylated histone H3 threonine‐3 (H3T3‐Phos), a mitotic histone “mark” appearing exclusively during cell division. Our approach identified survivin, the only known H3T3‐Phos‐binding protein, as well as other proteins, such as MCAK and KIF2A, that are likely to be involved in weak but selective interactions with this histone phosphorylation “mark”. Finally, we demonstrate that the CLASPI approach can be used to study the interplay between histone H3T3‐Phos and trimethylation on the adjacent residue lysine 4 (H3K4Me₃). Together, our findings indicate the CLASPI approach can be broadly applied to profile protein–protein interactions mediated by PTMs.     

Identification of new candidate drugs for lung cancer using chemical–chemical interactions,chemical–protein interactions and a K-means clustering algorithm

Lung cancer, characterized by uncontrolled cell growth in the lung tissue, is the leading cause of global cancer deaths. Until now, effective treatment of this disease is limited. Many synthetic compounds have emerged with the advancement of combinatorial chemistry. Identification of effective lung cancer candidate drug compounds among them is a great challenge. Thus, it is necessary to build effective computational methods that can assist us in selecting for potential lung cancer drug compounds. In this study, a computational method was proposed to tackle this problem. The chemical–chemical interactions and chemical–protein interactions were utilized to select candidate drug compounds that have close associations with approved lung cancer drugs and lung cancer-related genes. A permutation test and K-means clustering algorithm were employed to exclude candidate drugs with low possibilities to treat lung cancer. The final analysis suggests that the remaining drug compounds have potential anti-lung cancer activities and most of them have structural dissimilarity with approved drugs for lung cancer.     

Electrospray ionization mass spectrometry of soybean lipoxygenases: N-terminal acetylation, chemical modification, and solution conformation

Electrospray ionization mass spectrometry was used to examine both the covalent structure and solution conformation of the soybean lipoxygenases. The post-translational modifications of two lipoxgyenases were identified as N-terminal acetylations by tandem mass spectrometry of peptides generated by trypsin digestion. The N-terminal sequence suggests that the proteins were substrates for the plant homolog of the N-terminal acetyltransferase complex C in yeast. Analysis of samples of native lipoxygenase-3 produced ions corresponding within experimental error to the mass of the N-acetylated polypeptide and one iron atom. The precision of the measurements was within roughly 100 ppm for the 96,856 Da protein. This made it possible to detect the addition of a single oxygen atom to the enzyme in a chemical modification reaction with cumene hydroperoxide. The acid-induced denaturation of lipoxygenase-3, which was accompanied by nearly complete loss of catalytic activity, was observed below pH 3.5 with the appearance of ions in the mass spectrum derived from the apoprotein. There was no evidence for the loss of iron in the absence of unfolding. Solutions of lipoxygenase-3 incubated in 0.1M acetic acid produced ions with a novel charge state distribution suggesting a unique conformation. Circular dichroism measurements showed that the secondary structure features of the native protein were retained in the new conformation. Dynamic light scattering revealed that the new conformation was not a consequence of protein aggregation as the hydrodynamic radius of lipoxygenase-3 was significantly smaller in acetic acid solution than at pH 7.0. Remarkably, the enzyme incubated in acetic acid retained full catalytic activity.     

Annexin A2/P11 interaction: new insights into annexin A2 tetramer structure by chemical crosslinking, high-resolution mass spectrometry, and computational modeling

During the past few years, the structural analysis of proteins and protein complexes by chemical crosslinking and mass spectrometry has enjoyed increasing popularity. With this approach we have investigated the quaternary structure of the complex between annexin A2 and p11, which is involved in numerous cellular processes. Although high-resolution data are available for both interaction partners as well as for the complex between two p11 subunits and two annexin A2 N-terminal peptides, the structure of the complete annexin A2/p11 heterotetramer has not yet been solved at high resolution. Thus, the quaternary structure of the biologically relevant, membrane-bound annexin A2/p11 complex is still under discussion, while the existence of a heterotetramer or a heterooctamer is the prevailing opinion. We gained further insight into the spatial organization of the annexin A2/p11 heterotetramer by employing chemical crosslinking combined with high-resolution mass spectrometry. Furthermore, tandem mass spectrometry served as a tool for an exact localization of crosslinked amino acid residues and for a confirmation of crosslinked product assignment. On the basis of distance constraints from the crosslinking data we derived structural models of the annexin A2/p11 heterotetramer by computational docking with Rosetta. We propose an octameric model for the annexin A2/p11 complex, which exerts annexin A2 function. The proposed structure of the annexin A2/p11 octamer differs from so far suggested models and sheds new light into annexin A2/p11 interaction.     

Factors affecting the use of 13C(alpha) chemical shifts to determine, refine, and validate protein structures

Interest centers here on the analysis of two different, but related, phenomena that affect side-chain conformations and consequently 13C(alpha) chemical shifts and their applications to determine, refine, and validate protein structures. The first is whether 13C(alpha) chemical shifts, computed at the DFT level of approximation with charged residues is a better approximation of observed 13C(alpha) chemical shifts than those computed with neutral residues for proteins in solution. Accurate computation of 13C(alpha) chemical shifts requires a proper representation of the charges, which might not take on integral values. For this analysis, the charges for 139 conformations of the protein ubiquitin were determined by explicit consideration of protein binding equilibria, at a given pH, that is, by exploring the 2(xi) possible ionization states of the whole molecule, with xi being the number of ionizable groups. The results of this analysis, as revealed by the shielding/deshielding of the 13C(alpha) nucleus, indicated that: (i) there is a significant difference in the computed 13C(alpha) chemical shifts, between basic and acidic groups, as a function of the degree of charge of the side chain; (ii) this difference is attributed to the distance between the ionizable groups and the 13C(alpha) nucleus, which is shorter for the acidic Asp and Glu groups as compared with that for the basic Lys and Arg groups; and (iii) the use of neutral, rather than charged, basic and acidic groups is a better approximation of the observed 13C(alpha) chemical shifts of a protein in solution. The second is how side-chain flexibility influences computed 13C(alpha) chemical shifts in an additional set of ubiquitin conformations, in which the side chains are generated from an NMR-derived structure with the backbone conformation assumed to be fixed. The 13C(alpha) chemical shift of a given amino acid residue in a protein is determined, mainly, by its own backbone and side-chain torsional angles, independent of the neighboring residues; the conformation of a given residue itself, however, depends on the environment of this residue and, hence, on the whole protein structure. As a consequence, this analysis reveals the role and impact of an accurate side-chain computation in the determination and refinement of protein conformation. The results of this analysis are: (i) a lower error between computed and observed 13C(alpha) chemical shifts (by up to 3.7 ppm), was found for approximately 68% and approximately 63% of all ionizable residues and all non-Ala/Pro/Gly residues, respectively, in the additional set of conformations, compared with results for the model from which the set was derived; and (ii) all the additional conformations exhibit a lower root-mean-square-deviation (1.97 ppm < or = rmsd < or = 2.13 ppm), between computed and observed 13C(alpha) chemical shifts, than the rmsd (2.32 ppm) computed for the starting conformation from which this additional set was derived. As a validation test, an analysis of the additional set of ubiquitin conformations, comparing computed and observed values of both 13C(alpha) chemical shifts and chi(1) torsional angles (given by the vicinal coupling constants, 3J(N-Cgamma) and 3J(C'-Cgamma), is discussed.     

Electrostatic interactions play an essential role in the binding of oleic acid with α‐lactalbumin in the HAMLET‐like complex: A study using charge‐specific chemical modifications

H uman α ‐lactalbumin m ade le thal to t umor cells (HAMLET) and its analogs are partially unfolded protein‐oleic acid (OA) complexes that exhibit selective tumoricidal activity normally absent in the native protein itself. To understand the nature of the interaction between protein and OA moieties, charge‐specific chemical modifications of lysine side chains involving citraconylation, acetylation, and guanidination were employed and the biophysical and biological properties were probed. Upon converting the original positively‐charged lysine residues to negatively‐charged citraconyl or neutral acetyl groups, the binding of OA to protein was eliminated, as were any cytotoxic activities towards osteosarcoma cells. Retention of the positive charges by converting lysine residues to homoarginine groups (guanidination); however, yielded unchanged binding of OA to protein and identical tumoricidal activity to that displayed by the wild‐type α‐lactalbumin‐oleic acid complex. With the addition of OA, the wild‐type and guanidinated α‐lactalbumin proteins underwent substantial conformational changes, such as partial unfolding, loss of tertiary structure, but retention of secondary structure. In contrast, no significant conformational changes were observed in the citraconylated and acetylated α‐lactalbumins, most likely because of the absence of OA binding. These results suggest that electrostatic interactions between the positively‐charged basic groups on α‐lactalbumin and the negatively‐charged carboxylate groups on OA molecules play an essential role in the binding of OA to α‐lactalbumin and that these interactions appear to be as important as hydrophobic interactions. Proteins 2013. © 2012 Wiley Periodicals, Inc.