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Structural basis for the specific cotranslational incorporation of p-boronophenylalanine into biosynthetic proteins André Schiefner, Lea Nästle, Marietta Landgraf, Andreas J. Reichert, and Arne Skerra Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00171 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 18, 2018
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Biochemistry
Structural basis for the specific cotranslational incorporation of pboronophenylalanine into biosynthetic proteins André Schiefner*, Lea Nästle, Marietta Landgraf, Andreas J. Reichert and Arne Skerra* Lehrstuhl für Biologische Chemie, Technische Universität München, 85354 Freising (Weihenstephan), Germany Supporting Information Placeholder ABSTRACT: The site-specific incorporation of the
non-natural amino acid p-boronophenylalanine (Bpa) into recombinant proteins enables the development of novel carbohydrate-binding functions as well as bioorthogonal chemical modification. To this end, Bpa is genetically encoded by an amber stop codon and cotranslationally inserted into the recombinant polypeptide chain at the ribosome by means of an artificial aminoacyl-tRNA synthetase (aaRS) in combination with a compatible suppressor tRNA. We describe the crystal structure of an aaRS specific for Bpa, which had been engineered on the basis of the TyrRS from M. jannaschii, in complex with both Bpa and AMP. The bound substrates are observed in an orientation resembling the aminoacyl-AMP mixed anhydride intermediate and are engaged in a network of four hydrogen bonds that allows specific recognition of the boronate moiety by the aaRS. Key determinant of this interaction is the coplanar alignment of its Glu162 carboxylate group with Bpa, resulting in two hydrogen bonds with the boronic acid substituent. Our structural study elucidates how a small set of five side chain exchanges within the TyrRS active site can switch its substrate specificity to the hydrophilic amino acid Bpa, which stimulates the reprogramming of other aaRS to recruit useful nonnatural amino acids for next generation protein engineering.
Due to their mild Lewis acid character, combined with high chemical stability, boronic acids (which carry one alkyl/aryl substituent and two hydroxyl groups attached to the electron-deficient sp2hybrized boron atom) are attractive for a wide range of chemical reactions, including Suzuki crosscoupling,1 Diels-Alder reactions,2 copper-catalyzed alkylation3 and asymmetric reduction.4 Moreover, the ability of boronic acids to form reversible covalent complexes with cis-diols,5 amino alcohols6 as
well as certain amino acids7 has been exploited in the development of potent serine hydrolase inhibitors8 and also selective sugar-binding reagents, either based on small molecules9 or on engineered proteins.10 Furthermore, boronates can be applied as neutron capture reagents in tumor therapy.11 Unlike the structurally related carboxylic acids, boronic acids are not found in biological macromolecules. Thus, the site-directed incorporation of boronate-functionalized amino acids opens new avenues for biosynthetic protein modification, alternative purification strategies, drug delivery and, in particular, engineering of proteins with selective sugar-binding capabilities such as Borocalins.10 Genetic encoding of L-p-boronophenylalanine (Bpa) in E. coli has been enabled by combining an artificial amber suppressor tRNA with a tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii (MjTyrRS) that was reprogrammed for Bpa specificity.12 Here we report the X-ray structural analysis of the BpatRNA synthetase (BpaRS) both in its apo form and in complex with the non-natural amino acid and the cofactor AMP. The X-ray structure of the ternary complex BpaRS•Bpa/AMP was refined to a resolution of 1.6 Å (Table S1). BpaRS crystallized as a physiological homodimer, whose dimer axis corresponds to the crystallographic 2-fold axis with one BpaRS•Bpa/AMP complex in the asymmetric unit. The complex harbors a substrate/product (Bpa/AMP) mixture, in which the bound AMP apparently originated from the enzymatic conversion of ATP in the presence of Bpa and Mg2+ ions, but absence of tRNA cosubstrate, during a prolonged crystallization period (see below). All 306 residues of the BpaRS polypeptide plus 3 residues of the His6tag were resolved in the electron density. Most importantly, excellent electron density was observed for Bpa and AMP. According to their omit FO-FC electron densities and refined atomic B values, both
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ligands show full occupancy (Figure 1, Table S1), whereas no bound Mg2+ ions were observed. MjTyrRS comprises two domains that can be subdivided into five structural parts:13 (i) an N-terminal extension (residues 1-27), (ii) the Rossmann-fold (RF) type catalytic domain (residues 28-105 and 165196), (iii) the connective-polypeptide (CP1) segment within RF (residues 106-164),14 which is involved in dimer formation, and (iv) the KMSKS loop (residues 197-212)15 that links RF to (v) the C-terminal domain (residues 213-306), which recognizes the anticodonloop of the tRNA. Compared with the wild type enzyme, BpaRS carries five mutations, Y32S, L65A, H70M, D158S, L162E, which alter the substrate specificity from Tyr to Bpa,12 plus an additional substitution D286R for improved recognition of the CUA anticodon.13 Furthermore, the conservative mutation M6L was introduced in the present study to prevent internal translational initiation (cf. Material and Methods). Superposition of the BpaRS Cα atoms 28–196 (RF + CP1), which harbor the binding pockets for Bpa and the nucleotide cosubstrate (Figure 1B), with those of the previously crystallized MjTyrRS•tRNA/Tyr complex (PDB code 1J1U)13 revealed a very low average RMSD value of 0.45 Å. The only noteworthy backbone deviation of residues that line the amino acid (aa) substrate pocket is seen at the C-terminal end of helix α8 in the CP1 insertion:13 there, Glu162 is shifted by 0.9 Å toward the aa pocket, leading to a specific interaction with Bpa as described below. Compared to previously published structures of non-natural aa ligands – mostly having bulky hydrophobic side chains – in complex with mutated versions of MjTyrRS (see Table S2) Bpa is considerably more hydrophilic; thus, its recognition requires specific polar interactions, which are more challenging to implement by enzyme engineering. Indeed, Bpa is bound in the aa-substrate pocket via eight hydrogen bonds as well as several van der Waals (vdW) contacts from both RF and CP1. The ammonium and carboxylate groups of Bpa are positioned by four hydrogen bonds with Tyr151, Gln155 and Gln173, identical to Tyr in the native MjTyrRS•tRNA/Tyr complex (Figure 1C). Interestingly, selective recognition of Bpa is achieved by further four hydrogen bonds directed at the polar boronate group in its side chain. To this end, the side chain carboxylate of Glu162 is perfectly aligned with the boronic acid substituent in para position of the phenylalanine moiety and forms a double hydrogen bond with both of its hydroxyl groups. Ser32 and Gln109 form additional water-mediated hydro-
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gen bonds, each with one of the hydroxyl groups (Figure 1B). vdW contacts are mainly mediated by Gly34, Met70 and Gln155 to the phenyl ring.
Figure 1. Interactions of the substrates Bpa and AMP with BpaRS. (A) Omit FO-FC electron density contoured at 3 σ (green mesh) with superimposed ligands. Carbon atoms of Bpa and AMP are shown in gold and light blue, respectively, while heteroatoms are colored according to the CPK coloring scheme. (B) Hydrogenbonding pattern of Bpa (orange) and AMP (light blue) bound to BpaRS (black). Labels of mutated residues are underlined. Water molecules mediating hydrogen bonds between substrates and enzyme are depicted as filled circles (gray). (C) Comparison of the amino acid binding sites of BpaRS (left) and TyrRS (right). Residues forming hydrogen bonds with the bound aa as well as mutated residues are shown as sticks. Water molecules within the aa-substrate pocket are depicted as spheres.
Both volume and hydrophilicity of the aasubstrate pocket are increased in BpaRS compared to MjTyrRS. The Y32S and D158S substitutions abolish hydrogen bonding to the phenolic hydroxyl group of the natural substrate while the mutations 2
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Biochemistry
Y32S and L65A enlarge the pocket. This enables reorientation of the Bpa side chain compared to Tyr bound to the wild type enzyme (Figure 1C), which is necessary for optimal hydrogen bond formation with Glu162. The replacements Y32S and L65A also permit the coordination of three additional water molecules in the aa-substrate pocket, two of which mediate the aforementioned hydrogen bonds of Ser32 and Gln109 with Bpa (Figure 1B and C). The two other mutations, D158S and H70M, just provide vdW contacts with the boronate group and the phenyl ring, respectively. In this context, Ser158 seems to be an optimal residue as it can, like Asp in the wild type enzyme, form hydrogen bonds with the side chains of Tyr114 and Gln109 (outside the aasubstrate pocket) and at the same time provide more space – around the boronate group – for the longer side chain of the non-natural aa. In the original screen for Bpa-specific mutants of MjTyrRS, Ala was also observed at this position and appears to be similarly suitable,12 yet disrupting the hydrogen bond with Gln109 and Tyr114 but maintaining vdW contacts with Bpa. At position 32, Gly had been observed as an alternative substitution and also seems to be tolerable, even though the hydrogen bonds of Ser32 to Bpa and Glu162 would get lost (cf. Figure 1B). Notably, only Ala had been observed at position 65;12 there, any larger side chains would likely clash with Bpa. Finally, the exchange H70M increases the hydrophobicity and allows vdW contact formation with Bpa. While His was also observed at this position during the combinatorial engineering of BpaRS,12 Leu and Phe appear suitable, too. The shape of the aa-substrate pocket as well as the presence of 6 water molecules in addition to the bound Bpa suggest that even larger aa substrates could be accepted by BpaRS, especially if having planar shape. The ternary BpaRS•Bpa/AMP complex provides the first X-ray structure of MjTyrRS or its engineered versions with a bound nucleotide cofactor. With 2.5 Å, the phosphate group of AMP is in perfect hydrogen bonding distance to the Bpa carboxylate. In fact, the mutual arrangement of both moieties resembles the aminoacyl-AMP mixed anhydride intermediate which can also form in the absence of the cognate tRNA cosubstrate.16 This structural finding suggests that, after formation of the BpaRS•aminoacyl-AMP intermediate – from D/LBpa and ATP present in the crystallization setup (see Supporting Information) – spontaneous hydrolysis of the mixed anhydride took place (in the absence of the tRNA) prior to harvest of the crystals.
In principle, three scenarios can account for this hydrolysis: (i) the long period between crystallization setup and harvest, (ii) reorientation of the Bpa side chain in the aa-substrate pocket imposes a conformational strain that renders the anhydride bond more susceptible to hydrolysis (compared with conventional aminoacyl-AMP) or (iii) the crystal environment favors incorporation of the hydrolysis products. AMP is bound to BpaRS via 10 hydrogen bonds. As expected, most of these interactions are provided by RF, which forms 6 direct and 3 water-mediated hydrogen bonds (Figure 1B). In addition, a hydrogen bond between the main chain carbonyl of M205 in the KMSKS loop and the amino group of adenine seems to favor a distinct conformation of this loop. Structural superposition of BpaRS•Bpa/AMP with TyrRS of Plasmodium falciparum in complex with tyrosyl-AMP17 revealed 35 % identity among 305 equivalent Cα postions, resulting in an RMSD of 1.8 Å and very similar positioning of the ligands (Figure S1). In particular, the adenine moiety shows identical interaction with the main chain of the KMSKS loop, whereas in BpaRS•Bpa/AMP the phosphate is rotated away from the Bpa carboxylic group (Figure S1). Cα, amino group and carbonyl carbon of Bpa are displaced on average by 1.4 Å compared to the equivalent atoms of the Tyr moiety in PfTyrRS (in absence of an anhydride bond between Bpa and AMP).
Figure 2. Superposition of the BpaRS•Bpa/AMP complex (blue) with apo-BpaRS (yellow). Bpa and AMP are shown as ball-and-sticks. Conformational differences within the KMSKS loop region are illustrated in the 3
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close-up view. Hydrogen bond formation between AMP and Met205 is indicated by a dashed line.
The apo-form of BpaRS (apo-BpaRS) was refined to a resolution of 2.3 Å (Table S1). In this case, the asymmetric unit contains eight polypeptide chains, corresponding to four BpaRS homodimers. All eight monomers adopt a so-called apo-closed conformation (similar to the aa-bound conformation), which is characterized by a shift of helix pairs α4/α5 and α7/α8 toward the aa-substrate pocket.18 The different monomers show mutual RMSD values between 0.28 and 0.61 Å (for all 306 Cα positions). Superposition with the substrate complex described above revealed comparable RMSD values ranging from 0.48 to 0.72 Å. Most of the deviations result from small differences in the relative domain orientations. Notably, comparison of the ligand-binding region comprising RF and CP1 resulted in an RMSD of
