RESUMEN
The site-specific chemical conjugation of proteins, following synthesis with an expanded genetic code, promises to advance antibody-based technologies, including antibody drug conjugation and the creation of bispecific Fab dimers. The incorporation of non-natural amino acids into antibodies not only guarantees site specificity but also allows the use of bio-orthogonal chemistry. However, the efficiency of amino acid incorporation fluctuates significantly among different sites, thereby hampering the identification of useful conjugation sites. In this study, we applied the codon reassignment technology to achieve the robust and efficient synthesis of chemically functionalized antibodies containing Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) at defined positions. This lysine derivative has a bio-orthogonally reactive group at the end of a long side chain, enabling identification of multiple new positions in Fab-constant domains, allowing chemical conjugation with high efficiency. An X-ray crystallographic study of a Fab variant with o-Az-Z-Lys revealed high-level exposure of the azido group to solvent, with six of the identified positions subsequently used to engineer "Variabodies", a novel antibody format allowing various connections between two Fab molecules. Our findings indicated that some of the created Variabodies exhibited agonistic activity in cultured cells as opposed to the antagonistic nature of antibodies. These results showed that our approach greatly enhanced the availability of antibodies for chemical conjugation and might aid in the development of new therapeutic antibodies.
Asunto(s)
Anticuerpos/química , Anticuerpos/genética , Código Genético , Azidas/química , Línea Celular Tumoral , Química Clic , Codón/genética , Escherichia coli/genética , Humanos , Lisina/química , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Trastuzumab/química , Trastuzumab/genéticaRESUMEN
The N (1)-methyladenosine residue at position 58 of tRNA is found in the three domains of life, and contributes to the stability of the three-dimensional L-shaped tRNA structure. In thermophilic bacteria, this modification is important for thermal adaptation, and is catalyzed by the tRNA m(1)A58 methyltransferase TrmI, using S-adenosyl-L-methionine (AdoMet) as the methyl donor. We present the 2.2 Å crystal structure of TrmI from the extremely thermophilic bacterium Aquifex aeolicus, in complex with AdoMet. There are four molecules per asymmetric unit, and they form a tetramer. Based on a comparison of the AdoMet binding mode of A. aeolicus TrmI to those of the Thermus thermophilus and Pyrococcus abyssi TrmIs, we discuss their similarities and differences. Although the binding modes to the N6 amino group of the adenine moiety of AdoMet are similar, using the side chains of acidic residues as well as hydrogen bonds, the positions of the amino acid residues involved in binding are diverse among the TrmIs from A. aeolicus, T. thermophilus, and P. abyssi.
Asunto(s)
Aquifoliaceae/enzimología , Complejos Multiproteicos/ultraestructura , S-Adenosilmetionina/química , ARNt Metiltransferasas/química , ARNt Metiltransferasas/ultraestructura , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Enlace de Hidrógeno , Datos de Secuencia Molecular , Unión Proteica , Pyrococcus abyssi/enzimología , Alineación de Secuencia , Thermus thermophilus/enzimologíaRESUMEN
In thermophilic bacteria, specific 2-thiolation occurs on the conserved ribothymidine at position 54 (T54) in tRNAs, which is necessary for survival at high temperatures. T54 2-thiolation is achieved by the tRNA thiouridine synthetase TtuA and sulfur-carrier proteins. TtuA has five conserved CXXC/H motifs and the signature PP motif, and belongs to the TtcA family of tRNA 2-thiolation enzymes, for which there is currently no structural information. In this study, we determined the crystal structure of a TtuA homolog from the hyperthermophilic archeon Pyrococcus horikoshii at 2.1 Å resolution. The P. horikoshii TtuA forms a homodimer, and each subunit contains a catalytic domain and unique N- and C-terminal zinc fingers. The catalytic domain has much higher structural similarity to that of another tRNA modification enzyme, TilS (tRNA(Ile)2 lysidine synthetase), than to the other type of tRNA 2-thiolation enzyme, MnmA. Three conserved cysteine residues are clustered in the putative catalytic site, which is not present in TilS. An in vivo mutational analysis in the bacterium Thermus thermophilus demonstrated that the three conserved cysteine residues and the putative ATP-binding residues in the catalytic domain are important for the TtuA activity. A positively charged surface that includes the catalytic site and the two zinc fingers is likely to provide the tRNA-binding site.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Proteínas Bacterianas/química , Ligasas de Carbono-Azufre/química , Estructura Terciaria de Proteína , Thermus thermophilus/enzimología , Tiouridina/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/enzimología , Modelos Moleculares , MutaciónRESUMEN
The hypermodified nucleoside N(6)-threonylcarbamoyladenosine resides at position 37 of tRNA molecules bearing U at position 36 and maintains translational fidelity in the three kingdoms of life. The N(6)-threonylcarbamoyl moiety is composed of L-threonine and bicarbonate, and its synthesis was genetically shown to require YrdC/Sua5. YrdC/Sua5 binds to tRNA and ATP. In this study, we analyzed the L-threonine-binding mode of Sua5 from the archaeon Sulfolobus tokodaii. Isothermal titration calorimetry measurements revealed that S. tokodaii Sua5 binds L-threonine more strongly than L-serine and glycine. The Kd values of Sua5 for L-threonine and L-serine are 9.3 µM and 2.6 mM, respectively. We determined the crystal structure of S. tokodaii Sua5, complexed with AMPPNP and L-threonine, at 1.8 Å resolution. The L-threonine is bound next to AMPPNP in the same pocket of the N-terminal domain. Thr118 and two water molecules form hydrogen bonds with AMPPNP in a unique manner for adenine-specific recognition. The carboxyl group and the side-chain hydroxyl and methyl groups of L-threonine are buried deep in the pocket, whereas the amino group faces AMPPNP. The L-threonine is located in a suitable position to react together with ATP for the synthesis of N(6)-threonylcarbamoyladenosine.
Asunto(s)
Adenilil Imidodifosfato/química , Proteínas Arqueales/química , Proteínas de Unión al ARN/química , Sulfolobus/química , Treonina/química , Adenilil Imidodifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/metabolismo , Calorimetría , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Unión Proteica , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Treonina/metabolismo , Difracción de Rayos XRESUMEN
One of the modified nucleosides that frequently occurs in rRNAs and tRNAs is 5-methylcytidine (m5C). Escherichia coli Fmu/RsmB/RrmB is an S-adenosyl-L-methionine (AdoMet)-dependent methyltransferase that forms m5C967 in 16S rRNA. Fmu/RsmB/RrmB homologues exist not only in bacteria but also in archaea and eukarya and constitute a large orthologous group in the RNA:m5C methyltransferase family. In the present study, the crystal structure of a homologue of E. coli Fmu/RsmB/RrmB from the archaeon Pyrococcus horikoshii (PH0851) complexed with an AdoMet analogue was determined at 2.55 Å resolution. The structure and sequence of the C-terminal catalytic domain are highly conserved compared with those of E. coli Fmu/RsmB/RrmB. In contrast, the sequence of the N-terminal domain is negligibly conserved between the bacterial and archaeal subfamilies. Nevertheless, the N-terminal domains of PH0851 and E. coli Fmu/RsmB/RrmB are both α-helical and adopt a similar topology. Next to the AdoMet-binding site, a positively charged cleft is formed between the N- and C-terminal domains. This cleft is conserved in the archaeal PH0851 homologues and seems to be suitable for binding the RNA substrate.
Asunto(s)
Metiltransferasas/química , Pyrococcus horikoshii/enzimología , Secuencia de Aminoácidos , Escherichia coli/enzimología , Proteínas de Escherichia coli/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , ARN Ribosómico 16S/química , ARN Ribosómico 16S/metabolismo , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
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.
Asunto(s)
Aminoacil-ARNt Sintetasas , Euryarchaeota/genética , Código Genético/genética , Ingeniería de Proteínas/métodos , Aminoácidos/genética , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Sitios de Unión , Sistema Libre de Células , Euryarchaeota/enzimología , Fragmentos Fab de Inmunoglobulinas/genética , Modelos Moleculares , Trastuzumab/genéticaRESUMEN
In the bacterial genetic-code system, the codon AUA is decoded as isoleucine by tRNA(Ile)(2) with the lysidine residue at the wobble position. Lysidine is derived from cytidine, with ATP and L-lysine, by tRNA(Ile) lysidine synthetase (TilS), which is an N-type ATP pyrophosphatase. In this study, we determined the crystal structure of Aquifex aeolicus TilS, complexed with ATP, Mg2+, and L-lysine, at 2.5 A resolution. The presence of the TilS-specific subdomain causes the active site to have two separate gateways, a large hole and a narrow tunnel on the opposite side. ATP is bound inside the hole, and L-lysine is bound at the entrance of the tunnel. The conserved Asp36 in the PP-motif coordinates Mg2+. In these initial binding modes, the ATP, Mg2+, and L-lysine are held far apart from each other, but they seem to be brought together for the reaction upon cytidine binding, with putative structural changes of the complex.
Asunto(s)
Adenosina Trifosfato/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Lisina/metabolismo , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Aminoacil-ARNt Sintetasas/química , Lisina/química , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
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, NÉ-(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.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Lisina/análogos & derivados , Lisina/metabolismo , Aminoacil-ARNt Sintetasas/genética , Cristalografía por Rayos X , Escherichia coli , Código Genético/genética , Lisina/química , Lisina/genética , Methanosarcina/genética , Modelos Moleculares , Ingeniería de Proteínas/métodos , ARN de Transferencia/metabolismoRESUMEN
Tyrosyl-tRNA synthetase (TyrRS) catalyzes the tyrosylation of tRNA(Tyr) in a two-step reaction. TyrRS has the "HIGH" and "KMSKS" motifs, which play essential roles in the formation of the tyrosyl-adenylate from tyrosine and ATP. Here, we determined the crystal structures of Archaeoglobus fulgidus and Pyrococcus horikoshii TyrRSs in the l-tyrosine-bound form at 1.8A and 2.2A resolutions, respectively, and that of Aeropyrum pernix TyrRS in the substrate-free form at 2.2 A. The conformation of the KMSKS motif differs among the three TyrRSs. In the A.pernix TyrRS, the KMSKS loop conformation corresponds to the ATP-bound "closed" form. In contrast, the KMSKS loop of the P.horikoshii TyrRS forms a novel 3(10) helix, which appears to correspond to the "semi-closed" form. This conformation enlarges the entrance to the tyrosine-binding pocket, which facilitates the pyrophosphate ion release after the tyrosyl-adenylate formation, and probably is involved in the initial tRNA binding. The KMSSS loop of the A.fulgidus TyrRS is somewhat farther from the active site and is stabilized by hydrogen bonds. Based on the three structures, possible structural changes of the KMSKS motif during the tyrosine activation reaction are discussed. We suggest that the insertion sequence just before the KMSKS motif, which exists in some archaeal species, enhances the binding affinity of the TyrRS for its cognate tRNA. In addition, a non-proline cis peptide bond, which is involved in the tRNA binding, is conserved among the archaeal TyrRSs.
Asunto(s)
Aeropyrum/enzimología , Archaeoglobus fulgidus/enzimología , Pyrococcus horikoshii/enzimología , Tirosina-ARNt Ligasa/química , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Prolina/química , Conformación Proteica , Tirosina/análogos & derivados , Tirosina/químicaRESUMEN
Leukemia stem cells (LSCs) that survive conventional chemotherapy are thought to contribute to disease relapse, leading to poor long-term outcomes for patients with acute myeloid leukemia (AML). We previously identified a Src-family kinase (SFK) member, hematopoietic cell kinase (HCK), as a molecular target that is highly differentially expressed in human primary LSCs compared with human normal hematopoietic stem cells (HSCs). We performed a large-scale chemical library screen that integrated a high-throughput enzyme inhibition assay, in silico binding prediction, and crystal structure determination and found a candidate HCK inhibitor, RK-20449, a pyrrolo-pyrimidine derivative with an enzymatic IC50 (half maximal inhibitory concentration) in the subnanomolar range. A crystal structure revealed that RK-20449 bound the activation pocket of HCK. In vivo administration of RK-20449 to nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/IL2rg(null) mice engrafted with highly aggressive therapy-resistant AML significantly reduced human LSC and non-stem AML burden. By eliminating chemotherapy-resistant LSCs, RK-20449 may help to prevent relapse and lead to improved patient outcomes in AML.
Asunto(s)
Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Adulto , Anciano , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Trasplante de Médula Ósea , Cristalografía por Rayos X , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Hematopoyesis/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Masculino , Ratones , Persona de Mediana Edad , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Proteínas Proto-Oncogénicas c-hck/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-hck/química , Proteínas Proto-Oncogénicas c-hck/metabolismo , Pirimidinas/uso terapéutico , Pirroles/uso terapéutico , ARN Interferente Pequeño/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Células Tumorales Cultivadas , Adulto JovenRESUMEN
tRNA:m(5)C methyltransferase Trm4 generates the modified nucleotide 5-methylcytidine in archaeal and eukaryotic tRNA molecules, using S-adenosyl-l-methionine (AdoMet) as methyl donor. Most archaea and eukaryotes possess several Trm4 homologs, including those related to diseases, while the archaeon Methanocaldococcus jannaschii has only one gene encoding a Trm4 homolog, MJ0026. The recombinant MJ0026 protein catalyzed AdoMet-dependent methyltransferase activity on tRNA in vitro and was shown to be the M. jannaschii Trm4. We determined the crystal structures of the substrate-free M. jannaschii Trm4 and its complex with sinefungin at 1.27 A and 2.3 A resolutions, respectively. This AdoMet analog is bound in a negatively charged pocket near helix alpha8. This helix can adopt two different conformations, thereby controlling the entry of AdoMet into the active site. Adjacent to the sinefungin-bound pocket, highly conserved residues form a large, positively charged surface, which seems to be suitable for tRNA binding. The structure explains the roles of several conserved residues that were reportedly involved in the enzymatic activity or stability of Trm4p from the yeast Saccharomyces cerevisiae. We also discuss previous genetic and biochemical data on human NSUN2/hTrm4/Misu and archaeal PAB1947 methyltransferase, based on the structure of M. jannaschii Trm4.
Asunto(s)
Adenosina/análogos & derivados , Methanococcaceae/enzimología , ARNt Metiltransferasas/química , Adenosina/química , Aminoácidos , Antibacterianos/química , Proteínas Bacterianas/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Unión Proteica , Conformación Proteica , Proteínas de Saccharomyces cerevisiaeRESUMEN
tRNA precursors undergo a maturation process, involving nucleotide modifications and folding into the L-shaped tertiary structure. The N1-methylguanosine at position 37 (m1G37), 3' adjacent to the anticodon, is essential for translational fidelity and efficiency. In archaea and eukaryotes, Trm5 introduces the m1G37 modification into all tRNAs bearing G37. Here we report the crystal structures of archaeal Trm5 (aTrm5) in complex with tRNA(Leu) or tRNA(Cys). The D2-D3 domains of aTrm5 discover and modify G37, independently of the tRNA sequences. D1 is connected to D2-D3 through a flexible linker and is designed to recognize the shape of the tRNA outer corner, as a hallmark of the completed L shape formation. This interaction by D1 lowers the K(m) value for tRNA, enabling the D2-D3 catalysis. Thus, we propose that aTrm5 provides the tertiary structure checkpoint in tRNA maturation.
Asunto(s)
Anticodón/química , ARN de Transferencia/química , Alanina/química , Archaea , Catálisis , Codón , Cristalografía por Rayos X/métodos , Cisteína/química , Cinética , Modelos Moleculares , Conformación Molecular , Mutación , Conformación Proteica , Estructura Terciaria de Proteína , TemperaturaRESUMEN
The conserved cytidine residue at position 56 of tRNA contributes to the maintenance of the L-shaped tertiary structure. aTrm56 catalyzes the 2'-O-methylation of the cytidine residue in archaeal tRNA, using S-adenosyl-L-methionine. Based on the amino acid sequence, aTrm56 is the most distant member of the SpoU family. Here, we determined the crystal structure of Pyrococcus horikoshii aTrm56 complexed with S-adenosyl-L-methionine at 2.48 A resolution. aTrm56 consists of the SPOUT domain, which contains the characteristic deep trefoil knot, and a unique C-terminal beta-hairpin. aTrm56 forms a dimer. The S-adenosyl-L-methionine binding and dimerization of aTrm56 were similar to those of the other SpoU members. A structure-based sequence alignment revealed that aTrm56 conserves only motif II, among the four signature motifs. However, an essential Arg16 residue is located at a novel position within motif I. Biochemical assays showed that aTrm56 prefers the L-shaped tRNA to the lambda form as its substrate.
Asunto(s)
Citidina/análogos & derivados , Citidina/química , ARN de Archaea/química , ARN de Transferencia/química , ARNt Metiltransferasas/química , ARNt Metiltransferasas/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Metilación , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Pyrococcus horikoshii/enzimología , ARN de Archaea/genética , ARN de Archaea/metabolismo , ARN de Transferencia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo , Homología de Secuencia de Aminoácido , ARNt Metiltransferasas/metabolismoRESUMEN
The bacterial tRNA adenosine deaminase (TadA) generates inosine by deaminating the adenosine residue at the wobble position of tRNA(Arg-2). This modification is essential for the decoding system. In this study, we determined the crystal structure of Aquifex aeolicus TadA at a 1.8-A resolution. This is the first structure of a deaminase acting on tRNA. A. aeolicus TadA has an alpha/beta/alpha three-layered fold and forms a homodimer. The A. aeolicus TadA dimeric structure is completely different from the tetrameric structure of yeast CDD1, which deaminates mRNA and cytidine, but is similar to the dimeric structure of yeast cytosine deaminase. However, in the A. aeolicus TadA structure, the shapes of the C-terminal helix and the regions between the beta4 and beta5 strands are quite distinct from those of yeast cytosine deaminase and a large cavity is produced. This cavity contains many conserved amino acid residues that are likely to be involved in either catalysis or tRNA binding. We made a docking model of TadA with the tRNA anticodon stem loop.