Rational design of environmentally responsive antibodies with pH-sensing synthetic amino acids.

Antibodies are widely used as therapeutic agents to tackle various diseases. In the present study, to enhance their clinical values, we rationally designed pH-responsivity by exploiting the idiosyncratic protonation/deprotonation profiles of non-natural amino acids. 3-Nitro-L-tyrosine, 3-cyano-L-tyrosine, and 3, 5-halogenated-L-tyrosine, each with near neutral pKa, were thus incorporated into Fab fragments in place of tyrosines and other residues in the variable regions. Cell-based assays showed that these modifications achieved up to 140-fold tighter binding to antigens and several-fold tighter cytotoxicity to antigen-expressing cell at pH 6.0 than pH 7.4. The pH-dependent binding effect was retained in full-length antibodies. In silico structural analyses revealed electrostatic repulsion at neutral pH between antigens and antibodies or inside the antibody as the underlying mechanisms of the acid preference, and this finding increases the designability of pH-dependent antigen binding. The development of antibodies responsive to the microenvironments of diseased tissues will allow more disease-related antigens to be targeted in treatments, because of the reduced cross-reactivity toward healthy tissues.

Protein-Protein Interface Identification by Site-Specific Photo-Cross-linking/Cleavage in Mammalian Cells.

Identification of protein-protein interfaces is necessary for understanding and regulating biological events. Genetic code expansion enables site-specific photo-cross-linking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the cross-linked region still remains challenging. Our new protocol enables its identification by pre-installing a site-specific cleavage site, an α-hydroxy acid (Nε-allyloxycarbonyl-α-hydroxyl-L-lysine acid, AllocLys-OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α-hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N-terminus or C-terminus, the crosslinked site is located on within the target protein. A series of AllocLys-OH introductions narrows down the crosslinked region. This combination of site-specific crosslinking and cleavage promises to be useful for revealing binding interfaces and protein complex geometries. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Search for crosslinkable sites Basic Protocol 2: Site-specific photo-cross-linking/cleavage.

Genetic Code Expansion and a Photo-Cross-Linking Reaction Facilitate Ribosome Display Selections for Identifying a Wide Range of Affinity Peptides.

Cell-free molecular display techniques have been utilized to select various affinity peptides from peptide libraries. However, conventional techniques have difficulties associated with the translational termination through in-frame UAG stop codons and the amplification of non-specific peptides, which hinders the desirable selection of low-affinity peptides. To overcome these problems, we established a scheme for ribosome display selection of peptide epitopes bound to monoclonal antibodies and then applied genetic code expansion with synthetic X-tRNAUAG reprogramming of the UAG codons (X = Tyr, Trp, or p-benzoyl-l-phenylalanine (pBzo-Phe)) to the scheme. Based on the assessment of the efficiency of in vitro translation with X-tRNAUAG, we carried out ribosome display selection with genetic code expansion using Trp-tRNAUAG, and we verified that affinity peptides could be identified efficiently regardless of the presence of UAG codons in the peptide coding sequences. Additionally, after evaluating the photo-cross-linking reactions of pBzo-Phe-incorporated peptides, we performed ribosome display selection of low-affinity peptides in combination with genetic code expansion using pBzo-Phe-tRNAUAG and photo-irradiation. The results demonstrated that sub-micromolar low-affinity peptide epitopes could be identified through the formation of photo-induced covalent bonds with monoclonal antibodies. Thus, the developed ribosome display techniques could contribute to the promotion of diverse peptide-based research.

Site-specific photo-crosslinking/cleavage for protein-protein interface identification reveals oligomeric assembly of lysosomal-associated membrane protein type 2A in mammalian cells.

Genetic code expansion enables site-specific photo-crosslinking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the crosslinked region still remains challenging. Here, we developed a new method to identify the crosslinked region by pre-installing a site-specific cleavage site, an α-hydroxy acid (Nε -allyloxycarbonyl-α-hydroxyl-l-lysine acid, AllocLys-OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α-hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N-terminus or C-terminus, the crosslinked site is located within the target protein. A series of AllocLys-OH introductions narrows down the crosslinked region. By applying this method, we identified the crosslinked regions in lysosomal-associated membrane protein type 2A (LAMP2A), a receptor of chaperone-mediated autophagy, in mammalian cells. The results suggested that at least two interfaces are involved in the homophilic interaction, which requires a trimeric or higher oligomeric assembly of adjacent LAMP2A molecules. Thus, the combination of site-specific crosslinking and site-specific cleavage promises to be useful for revealing binding interfaces and protein complex geometries.

Crystal Structure of Pyrrolysyl-tRNA Synthetase from a Methanogenic Archaeon ISO4-G1 and Its Structure-Based Engineering for Highly-Productive Cell-Free Genetic Code Expansion with Non-Canonical Amino Acids.

Pairs of pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl from Methanosarcina mazei and Methanosarcina barkeri are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). Previously, we achieved full productivity of cell-free protein synthesis for bulky non-canonical amino acids, including Nε-((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS with structure-based mutations in and around the amino acid binding pocket (first-layer and second-layer mutations, respectively). Recently, the PylRS·tRNAPyl pair from a methanogenic archaeon ISO4-G1 was used for genetic code expansion. In the present study, we determined the crystal structure of the methanogenic archaeon ISO4-G1 PylRS (ISO4-G1 PylRS) and compared it with those of structure-known PylRSs. Based on the ISO4-G1 PylRS structure, we attempted the site-specific incorporation of Nε-(p-ethynylbenzyloxycarbonyl)-L-lysine (pEtZLys) into proteins, but it was much less efficient than that of TCO*Lys with M. alvus PylRS mutants. Thus, the first-layer mutations (Y125A and M128L) of ISO4-G1 PylRS, with no additional second-layer mutations, increased the protein productivity with pEtZLys up to 57 ± 8% of that with TCO*Lys at high enzyme concentrations in the cell-free protein synthesis.

Genetic Code Expansion: Another Solution to Codon Assignments.

This Special Issue is intended to highlight recent advances in genetic code expansion, particularly the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins [...].

The two-domain architecture of LAMP2A regulates its interaction with Hsc70.

Chaperone-mediated autophagy (CMA) is a unique proteolytic pathway, in which cytoplasmic proteins recognized by heat shock cognate protein 70 (Hsc70/HSPA8) are transported into lysosomes for degradation. The substrate/chaperone complex binds to the cytosolic tail of the lysosomal-associated membrane protein type 2A (LAMP2A), but whether the interaction between Hsc70 and LAMP2A is direct or mediated by other molecules has remained to be elucidated. The structure of LAMP2A comprises a large lumenal domain composed of two domains, both with the ß-prism fold, a transmembrane domain and a short cytoplasmic tail. We previously reported the structural basis for the homophilic interaction of the lumenal domains of LAMP2A, using site-specific photo-crosslinking and/or steric hindrance within cells. In the present study, we introduced a photo-crosslinker into the cytoplasmic tail of LAMP2A and successfully detected its crosslinking with Hsc70, revealing this direct interaction for the first time. Furthermore, we demonstrated that the truncation of the membrane-distal domain within the lumenal domain of LAMP2A reduced the amount of Hsc70 that coimmunoprecipitated with LAMP2A. Our present results suggested that the two-domain architecture of the lumenal domains of LAMP2A underlies the interaction with Hsc70 at the cytoplasmic surface of the lysosome.

An integrated computational pipeline for designing high-affinity nanobodies with expanded genetic codes.

Protein engineering and design principles employing the 20 standard amino acids have been extensively used to achieve stable protein scaffolds and deliver their specific activities. Although this confers some advantages, it often restricts the sequence, chemical space, and ultimately the functional diversity of proteins. Moreover, although site-specific incorporation of non-natural amino acids (nnAAs) has been proven to be a valuable strategy in protein engineering and therapeutics development, its utility in the affinity-maturation of nanobodies is not fully explored. Besides, current experimental methods do not routinely employ nnAAs due to their enormous library size and infinite combinations. To address this, we have developed an integrated computational pipeline employing structure-based protein design methodologies, molecular dynamics simulations and free energy calculations, for the binding affinity prediction of an nnAA-incorporated nanobody toward its target and selection of potent binders. We show that by incorporating halogenated tyrosines, the affinity of 9G8 nanobody can be improved toward epidermal growth factor receptor (EGFR), a crucial cancer target. Surface plasmon resonance (SPR) assays showed that the binding of several 3-chloro-l-tyrosine (3MY)-incorporated nanobodies were improved up to 6-fold into a picomolar range, and the computationally estimated binding affinities shared a Pearson's r of 0.87 with SPR results. The improved affinity was found to be due to enhanced van der Waals interactions of key 3MY-proximate nanobody residues with EGFR, and an overall increase in the nanobody's structural stability. In conclusion, we show that our method can facilitate screening large libraries and predict potent site-specific nnAA-incorporated nanobody binders against crucial disease-targets.

Direct homophilic interaction of LAMP2A with the two-domain architecture revealed by site-directed photo-crosslinks and steric hindrances in mammalian cells.

LAMP1 (lysosomal-associated membrane protein 1) and LAMP2 are the most abundant protein components of lysosome membranes. Both LAMPs have common structures consisting of a large lumenal domain composed of two domains (N-domain and C-domain, which are membrane-distal and -proximal, respectively), both with the ß-prism fold, a transmembrane domain, and a short cytoplasmic tail. LAMP2 is involved in various aspects of autophagy, and reportedly forms high-molecular weight complexes at the lysosomal membrane. We previously showed that LAMP2 molecules coimmunoprecipitated with each other, but whether the homophilic interaction is direct or indirect has remained to be elucidated. In the present study, we demonstrated the direct homophilic interaction of mouse LAMP2A molecules, using expanded genetic code technologies that generate photo-crosslinking and/or steric hindrance at specified interfaces. Specifically, the results suggested that LAMP2A molecules assemble by facing each other with one side of the ß-prism (defined as side A) of the C-domains. The N-domain truncation, which increased the coimmunoprecipitation of LAMP2A molecules in our previous study, permitted the nonspecific involvement of both sides of the ß-prism (side A and side B). Thus, the presence of the N-domain restricts the LAMP2A interactions to side A-specific. The truncation of LAMP2A impaired the recruitment of GAPDH (a CMA-substrate) fused to the HaloTag protein to the surface of late endosomes/lysosomes (LE/Lys) and affected a process that generates LE/Lys. The present study revealed that the homophilic interaction of LAMP2A is direct, and the side A-specific, homophilic interaction of LAMP2A is required for the functional aspects of LAMP2A.Abbreviations: Aloc-Lys: Nε-allyloxycarbonyl-l-lysine; CMA: chaperone-mediated autophagy; FFE: free-flow electrophoresis; GAPDH-HT: glyceraldehyde-3-phosphate dehydrogenase fused to HaloTag protein; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LBPA: lysobisphosphatidic acid; LE/Lys: late endosome/lysosomes; MEFs: mouse embryonic fibroblasts; pBpa: p-benzoyl- l-phenylalanine.

An expanded genetic code facilitates antibody chemical conjugation involving the lambda light chain.

Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o-Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical conjugation method to the whole spectrum of humanized antibodies, including the λ isotype.

Simulation code for estimating external gamma-ray doses from a radioactive plume and contaminated ground using a local-scale atmospheric dispersion model.

In this study, we developed a simulation code powered by lattice dose-response functions (hereinafter SIBYL), which helps in the quick and accurate estimation of external gamma-ray doses emitted from a radioactive plume and contaminated ground. SIBYL couples with atmospheric dispersion models and calculates gamma-ray dose distributions inside a target area based on a map of activity concentrations using pre-evaluated dose-response functions. Moreover, SIBYL considers radiation shielding due to obstructions such as buildings. To examine the reliability of SIBYL, we investigated five typical cases for steady-state and unsteady-state plume dispersions by coupling the Gaussian plume model and the local-scale high-resolution atmospheric dispersion model using large eddy simulation. The results of this coupled model were compared with those of full Monte Carlo simulations using the particle and heavy-ion transport code system (PHITS). The dose-distribution maps calculated using SIBYL differed by up to 10% from those calculated using PHITS in most target locations. The exceptions were locations far from the radioactive contamination and those behind the intricate structures of building arrays. In addition, SIBYL's computation time using 96 parallel processing elements was several tens of minutes even for the most computationally expensive tasks of this study. The computation using SIBYL was approximately 100 times faster than the same calculation using PHITS under the same computation conditions. From the results of the case studies, we concluded that SIBYL can estimate a ground-level dose-distribution map within one hour with accuracy that is comparable to that of the full Monte Carlo simulation.

Incorporation of Halogenated Amino Acids into Antibody Fragments at Multiple Specific Sites Enhances Antigen Binding.

Expansion of the amino-acid repertoire with synthetic derivatives introduces novel structures and functionalities into proteins. In this study, we improved the antigen binding of antibodies by incorporating halogenated tyrosines at multiple selective sites. Tyrosines in the Fab fragment of an anti-EGF-receptor antibody 059-152 were systematically replaced with 3-bromo- and 3-chlorotyrosines, and simultaneous replacements at four specific sites were found to cause a tenfold increase in the affinity toward the antigen. Structure modeling suggested that this effect was due to enhanced shape complementarity between the antigen and antibody molecules. On the other hand, we showed that chlorination in the constant domain, far from the binding interface, of Rituximab Fab also increased the affinity significantly (up to 17-fold). Our results showed that antigen binding is tunable with the halogenation in and out of the binding motifs.

Fully Productive Cell-Free Genetic Code Expansion by Structure-Based Engineering of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase.

Pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs from Methanosarcina mazei and Methanosarcina barkeri are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). In this study, we achieved the full productivity of cell-free protein synthesis for difficult, bulky non-canonical amino acids, such as Nε-((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)-l-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS. First, based on the crystal structure of M. alvus PylRS, the productivities for various non-canonical amino acids were greatly increased by rational engineering of the amino acid-binding pocket. The productivities were further enhanced by using a much higher concentration of PylRS over that of M. mazei PylRS, or by mutating the outer layer of the amino acid-binding pocket. Thus, we achieved full productivity even for TCO*Lys. The quantity and quality of the cell-free-produced antibody fragment containing TCO*Lys were drastically improved. These results demonstrate the importance of full productivity for the expanded genetic code.

Structural Basis for Genetic-Code Expansion with Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase.

Pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl have been extensively used for genetic-code expansion. A Methanosarcina mazei PylRS mutant bearing the Y306A and Y384F mutations (PylRS(Y306A/Y384F)) encodes various bulky non-natural lysine derivatives by UAG. In this study, we examined how PylRS(Y306A/Y384F) recognizes many amino acids. Among 17 non-natural lysine derivatives, Nɛ-(benzyloxycarbonyl)lysine (ZLys) and 10 ortho/meta/para-substituted ZLys derivatives were efficiently ligated to tRNAPyl and were incorporated into proteins by PylRS(Y306A/Y384F). We determined crystal structures of 14 non-natural lysine derivatives bound to the PylRS(Y306A/Y384F) catalytic fragment. The meta- and para-substituted ZLys derivatives are snugly accommodated in the productive mode. In contrast, ZLys and the unsubstituted or ortho-substituted ZLys derivatives exhibited an alternative binding mode in addition to the productive mode. PylRS(Y306A/Y384F) displayed a high aminoacylation rate for ZLys, indicating that the double-binding mode minimally affects aminoacylation. These precise substrate recognition mechanisms by PylRS(Y306A/Y384F) may facilitate the structure-based design of novel non-natural amino acids.

Cell-Free Protein Synthesis Using S30 Extracts from Escherichia coli RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins.

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.

Variants of the industrially relevant protease KP-43 with suppressed activity under alkaline conditions developed using expanded genetic codes.

In the present study, we attempted to control the pH profile of the catalytic activity of the industrially relevant alkaline protease KP-43, by incorporating 3-nitro-l-tyrosine and 3-chloro-l-tyrosine at and near the catalytic site. Thirty KP-43 variants containing these non-natural amino acids at the specific positions were synthesized in Escherichia coli host cells with expanded genetic codes. The variant with 3-nitrotyrosine at position 205, near the substrate binding site, retained its catalytic activity at the neutral pH and showed a 60% activity reduction at pH 10.5. This reduction in the alkaline domain is desirable for enhancing the stability of the enzyme in the liquid laundary detergent, whereas the wild-type molecule showed a 20% increase in response to the same pH shift. The engineered pH dependency of the activity of the variant was ascribed partly to a lowered substrate affinity under the alkaline conditions, in which the incorporated 3-nitrotyrosine was probably charged negatively due to the phenolic pK a lower than that of tyrosine.

Synthetic Tyrosine tRNA Molecules with Noncanonical Secondary Structures.

The L-shape form of tRNA is maintained by tertiary interactions occurring in the core. Base changes in this domain can cause structural defects and impair tRNA activity. Here, we report on a method to safely engineer structural variations in this domain utilizing the noncanonical scaffold of tRNAPyl. First, we constructed a naïve hybrid between archaeal tRNAPyl and tRNATyr, which consisted of the acceptor and T stems of tRNATyr and the other parts of tRNAPyl. This hybrid tRNA efficiently translated the UAG codon to 3-iodotyrosine in Escherichia coli cells, when paired with a variant of the archaeal tyrosyl-tRNA synthetase. The amber suppression efficiency was slightly lower than that of the "bench-mark" archaeal tRNATyr suppressor assuming the canonical structure. After a series of modifications to this hybrid tRNA, we obtained two artificial types of tRNATyr: ZtRNA had an augmented D (auD) helix in a noncanonical form and the D and T loops bound by the standard tertiary base pairs, and YtRNA had a canonical auD helix and non-standard interloop interactions. It was then suggested that the ZtRNA scaffold could also support the glycylation and glutaminylation of tRNA. The synthetic diversity of tRNA would help create new tRNA⁻aminoacyl-tRNA synthetase pairs for reprogramming the genetic code.

Engineering of Escherichia coli ß-lactamase TEM-1 variants showing higher activity under acidic conditions than at the neutral pH.

Escherichia coli ß-lactamase TEM-1 is potentially useful in the antibody-directed enzyme/prodrug therapy (ADEPT), converting nontoxic prodrugs to toxic agents. The produced toxin would kill cancer cells, when the enzyme is attached to a tumor-antigen-specific antibody. However, the off-site reaction possibly occurring in the blood or normal tissues raises safety concern. In the present study, we engineered TEM-1 variants preferentially active at pH 5.8-6.2, near the pH of the acidic microenvironment of tumor. A library of randomly mutagenized variants was screened for the ability to confer an antibiotic resistance on E. coli cells in acidic growth media and not in neutral media, to isolate a variant with a Thr-to-Ile substitution at position 160. An extensive mutagenesis study was then conducted in the proximity of this position, to show that a Leu162Glu mutation also causes the acid preference. Kinetic analyses indicated that the overall activity of the wild-type TEM-1 hardly changes over a pH range from 5.8 to 7.0, whereas TEM-1(T160I) is 1.5-times as active at pH 6.2 than pH 7.0, and TEM-1(T160I) is 3.1-fold as active at pH 5.8 than pH 7.0. A further mutagenesis study suggested that a change in the overall structure of the enzyme underlies the pH dependency of the variants.

Pyrrolysyl-tRNA Synthetase with a Unique Architecture Enhances the Availability of Lysine Derivatives in Synthetic Genetic Codes.

Genetic code expansion has largely relied on two types of the tRNA-aminoacyl-tRNA synthetase pairs. One involves pyrrolysyl-tRNA synthetase (PylRS), which is used to incorporate various lysine derivatives into proteins. The widely used PylRS from Methanosarcinaceae comprises two distinct domains while the bacterial molecules consist of two separate polypeptides. The recently identified PylRS from Candidatus Methanomethylophilus alvus (CMaPylRS) is a single-domain, one-polypeptide enzyme that belongs to a third category. In the present study, we showed that the PylRS-tRNAPyl pair from C. M. alvus can incorporate lysine derivatives much more efficiently (up to 14-times) than Methanosarcinaceae PylRSs in Escherichia coli cell-based and cell-free systems. Then we investigated the tRNA and amino-acid recognition by CMaPylRS. The cognate tRNAPyl has two structural idiosyncrasies: no connecting nucleotide between the acceptor and D stems and an additional nucleotide in the anticodon stem and it was found that these features are hardly recognized by CMaPylRS. Lastly, the Tyr126Ala and Met129Leu substitutions at the amino-acid binding pocket were shown to allow CMaPylRS to recognize various derivatives of the bulky Nε-benzyloxycarbonyl-l-lysine (ZLys). With the high incorporation efficiency and the amenability to engineering, CMaPylRS would enhance the availability of lysine derivatives in expanded codes.