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Title:
Designed Enzyme Promotes Selective Post-translational Acylation

Paper sections:

<p>Post-translational modifications (PTM) play a major role in modulating protein activity, protein-protein interactions, subcellular localization and DNA binding. Acylation of lysine residues is commonly observed in various proteins highlighting its broad regulatory function in diverse cellular processes.[1] Acyltransferases and deacylases modify the core histone tails and play a critical role in gene expression.[2] To decipher the role that lysine acylation plays in biological function, it is essential to develop tools that introduce this modification at a particular residue. Several successful approaches for site specific introduction of acyl group have been developed, but the currently available methods do have their limitations. Enzymatic acylation[3] by purified acyltransferases can give rise to heterogeneous samples with incomplete or multiple acylation sites.[4] While the existing methods for incorporation of post-translational modifications can be quite successful[5] the ever expanding needs of chemical biology require additional approaches to site-specific introduction of an acyl group.</p><p>Recently we demonstrated that strategic introduction of a single mutation into C-terminal domain of calmodulin (c-CaM) confers esterase activity onto this non-enzymatic protein (Figure 1). The resulting minimalist stereoselective esterase, AlleyCatE, is highly active and allosterically regulated. Over the course of AlleyCatE-promoted hydrolysis of p-nitrophenyl esters, multiple self-acylation events were observed in addition to normal substrate turnover.[6] Given remarkable ability of calmodulin to bind a diverse set of proteins with high affinity, AlleyCatE's efficient transfer of acyl group presents an opportunity to selectively modify specific functional groups.</p><p>Calmodulin (CaM) is one of the most abundant eukaryotic proteins that responds to changes in local calcium concentration to modulate activities of a variety of protein kinases, phosphodiesterases, calcium pumps, etc.[7] Binding of four calcium ions to CaM induces a conformational change which enables it to recognize and bind protein targets. Amazingly, despite large variability in primary sequence of calmodulin-binding proteins, CaM binds them with high (in many cases nM) affinity. CaM's low apparent selectivity and high affinity for many targets is a result of its ability to recognize patterns of charged and hydrophobic residues and to adopt multiple binding modes and stoichiometries.[8] The well-characterized interaction of calmodulin-dependent kinase II (CaMKII) with CaM provides an important insight into how calmodulin binds many of its targets: the two domains of CaM engulf the helical region of CaMKII (Figure 1). Given the structural similarity of CaM and AlleyCatE we set out to explore the possibility of selective acyl transfer from the active His144 residue of the designed esterase onto the CaM-binding domain of CaMKII.</p><p>To test the possibility of an acyl transfer we chose CaM M144H, a variant of AlleyCatE which includes both N-terminal and C-terminal domains, and a well-characterized fragment of CaMKII from 294 to 314 (Table 1),[9] with nM affinity for CaM. Structural alignment of the C-terminal domain of calmodulin with AlleyCatE (Figure 1) shows that the active site histidine residue of the esterase is located close to Lys300 of the CaMKII294–314; the side chain of other lysine residue Lys298 points in the opposite direction.</p><p>Armed with this structural prediction we tested acylation of CaMKII294–314 by p-nitrophenyl-(2-phenyl)-propanoate (pNPP), a substrate that provides a convenient mass spectrometric marker and that was previously shown to be effectively activated by AlleyCatE.[6] Direct addition of pNPP to CaMKII294–314 in the absence of CaM M144H does not result in any acyl transfer. However, when calcium-bound CaM M144H is present, a high degree of peptide acylation is evident from MALDI-TOF spectra and HPLC chromatogram in as little as 15 minutes (Figure 2, Figures S1-S2, Supporting Information), reaching nearly stoichiometric conversion in 6 hours (Figure S1) and a Vmax/[E] of 0.24 min−1. The acyl transfer can be achieved in a broad range of calcium concentrations reaching as low as 10 μM (Figure S3). To maximize the yield the reactions have been conducted under nearly stoichiometric conditions (peptide:pNPP=1:2). Importantly, MS-MS analysis demonstrated that the acylation pattern is consistent with the phenylpropionate group attached to Lys300 in complete agreement with the structural model (Figure S4). The acyl transfer is very efficient – the ratio of the acylated product to the overall hydrolysis (including the background uncatalyzed rate) is 1:2 (Figure S1).</p><p>Allosterically regulated proteins provide a convenient opportunity to exclude any adventitious interaction contributing to catalysis by removing the allosteric modulator (calcium ions in case of CaM M144H). This is a very stringent test[10] that allows for probing of the reaction mechanism as the removal of calcium from EF-hands of CaM M144H results in a catalytically inactive conformation. To ensure that the observed acyl transfer is due to the designed protein we performed the reaction in the presence of both CaM M144H and EDTA. No acylation was observed once calcium was effectively complexed even though CaM M144H was present in the reaction mixture (Figure 2).</p><p>Previously[6] we have established that AlleyCatE shows selectivity for pNPP over p-nitrophenyl acetate (pNPA), thus we set out to explore whether CaM M144H possesses similar selectivity in acyl transfer reactions. The transfer of acetate by CaM 144H onto CaMKII294–314 is very slow and is barely above the background rate (Figure S5).</p><p>AlleyCatE undergoes self-acylation over the prolonged period of time. Interestingly the self-acylation profile of CaM M144H over 24 h is essentially the same than that of AlleyCat (Figures S6, S7), however CaM M144H is self-acylated much slower. As expected, in the presence of CaMKII294–314 the self-acylation is inhibited even further (Figure S8). In order to determine the specific acylation sites we performed tryptic digest followed by MS-MS analysis of the fragments. Essentially all of the residues modified (with an exception of Lys13) are lysine residues located in the C-terminal domain – Lys75, Lys77, Lys94 and Lys148 (Figure S9).</p><p>To investigate whether CaM M144H is capable of acyl transfer onto other peptides that bind CaM we looked at phosphofructokinase (PFK) that plays a key role in glycolysis regulation.[11] The calmodulin-binding region of PFK is very different from that of CaMKII in terms of primary sequence (Table 1), but it nonetheless has very high affinity for CaM (11.4 nM).[9b] No structural information for the CaM-PFK interaction has been reported, so successful acylation of PFK by CaM M144H will not only demonstrate generality of CaM M144H -promoted acylation, but will show it to be a chemical biology tool for mapping protein-protein interactions.</p><p>We chose the peptide that corresponds to a region between residues 378 to 403 of PFK (Table 1). The resulting PFK kinase peptide (PFK378–403) shows about an order of magnitude lower affinity for CaM as compared to CaMKII294–314. No acylation of PFK378–403 is evident upon addition of pNPP or in the presence of CaM M144H and EDTA. In the presence of calcium, however, the peptide is acylated albeit at a slower rate than CaMKII294–314 (Figures S10-S12). The MS2 analysis of the product shows that the peptide is selectively acylated at position 386 with only a minor potential acylation at the other position (Figure S13).</p><p>Selective acylation of PFK378–403 in position 386 by CaM M144H suggests physical proximity of Met144 of CaM and Lys386 of PFK as the binding mode of the peptide is unlikely to be drastically altered by a single mutation. We used this experimentally derived constraint to computationally predict and validate the structure of CaM-PFK378–403 complex. As a starting point we took a structure of CaM from the CaM-CaMKII294–314 complex, (PDB code 3GP2) which represents a very common mode of CaM interactions with its targets, and a fragment of a crystal structure of rabbit muscle PFK with primary sequence identical to the PFK378–403 peptide used in this study (PDB code 3O8L).[12] Computational docking of CaM and the PFK378–403 fragment with flexible side chains using Rosetta provides a model that is fully consistent with our acylation studies (Figure 3). In the pose with the lowest Rosetta score, Lys386 is located in proximity to Met144, whereas Lys397 is found on the other side of CaM, far away from the acylation site.</p><p>To establish whether our methodology can be applied to large proteins we recombinantly expressed full length CaMKII, a 475 residue protein that forms a large homododecameric assembly of more than 0.5 MDa in size. No structural information is currently available for CaM-full length CaMKII complex. LC-MS2 analysis of the tryptic digest of CaMKII incubated with pNPP for 20 hours shows no signs of (2-phenyl)-propanoate transfer onto the protein, however when the reaction was done in the presence of calcium-bound CaM M144H acylation of CaMKII was evident (Tables S1, S2). Unfortunately, due to the large size of the protein and uneven pattern of tryptic digestion we lacked full mass spectroscopic coverage of the sequence (reaching at most 50%), including that for the known CaM binding domain. The observed acylation site is located in the αC helix of CaMKII and it is feasible that it is in close proximity to the CaM-binding domain of the protein upon the conformational change induced by CaM binding. Future studies will be necessary to elucidate the details of this interaction, but there is every reason to believe that CaM M144H will serve as a very useful tool in studies of CaMKII activation.</p><p>Minimalist design of enzymes has been very fruitful in creating efficient catalysts at very little computational and experimental cost.[6, 10b, 13] We have demonstrated that an allosterically regulated catalyst produced by substituting a single residue in a non-enzymatic protein is capable of selective post- translational modification of peptides with highly diverse sequences. Substrate selectivity is often minimal in de novo designed or redesigned proteins. To our knowledge this is the first demonstration of selective modification of a complex target by a computationally designed protein. By utilizing unique recognition properties of calmodulin and very basic physico-chemical principles we were able to label CaM-binding partners with an unnatural tag that can be easily identified through mass spectrometry. While this approach is by no means applicable to all proteins/peptides, and a much larger study will be needed to test the limits of its applicability in complex biological systems we envision CaM M144H to become a valuable chemical biology tool to determine the calmodulin-binding domains. This is a very important, considering involvement of CaM in various biological pathways, but often difficult task given the transient nature of such interactions. Moreover, acyl transfer results provide distance constraints helpful for elucidation of structural modes of CaM-protein interactions. Although insertion of cross-linkers using amber codon suppression or protein semi-synthesis can provide similar insight,[14] no complex genetic modifications or chemical synthesis is required in our approach.</p>
A computationally designed allosterically regulated catalyst (CaM M144H) produced by substituting a single residue in calmodulin, a non-enzymatic protein, is capable of efficient and site selective post-translational acylation of lysines in peptides with highly diverse sequences. Calmodulin binding partners are involved in regulating a large number of cellular processes, this new chemical biology tool will help identify them and provide structural insight into their interactions with calmodulin.