RESUMO
A distinctive feature of human IgG4 is its ability to recombine half molecules (H chain and attached L chain) through a dynamic process termed Fab-arm exchange, which results in bispecific Abs. It is becoming evident that the process of Fab-arm exchange is conserved in several mammalian species, and thereby represents a mechanism that impacts humoral immunity more generally than previously thought. In humans, Fab-arm exchange has been attributed to the IgG4 core-hinge sequence (226-CPSCP-230) in combination with unknown determinants in the third constant H chain domain (CH3). In this study, we investigated the role of the CH3 domain in the mechanism of Fab-arm exchange, and thus identified amino acid position 409 as the critical CH3 determinant in human IgG, with R409 resulting in exchange and K409 resulting in stable IgG. Interestingly, studies with IgG from various species showed that Fab-arm exchange could not be assigned to a common CH3 domain amino acid motif. Accordingly, in rhesus monkeys (Macaca mulatta), aa 405 was identified as the CH3 determinant responsible (in combination with 226-CPACP-230). Using native mass spectrometry, we demonstrated that the ability to exchange Fab-arms correlated with the CH3-CH3 dissociation constant. Species-specific adaptations in the CH3 domain thus enable Fab-arm exchange by affecting the inter-CH3 domain interaction strength. The redistribution of Ag-binding domains between molecules may constitute a general immunological and evolutionary advantage. The current insights impact our view of humoral immunity and should furthermore be considered in the design and evaluation of Ab-based studies and therapeutics.
Assuntos
Anticorpos Biespecíficos/química , Imunoglobulina G/química , Cadeias Pesadas de Imunoglobulinas/química , Modelos Moleculares , Animais , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Fragmentos Fab das Imunoglobulinas/química , Macaca mulatta , Espectrometria de Massas , Especificidade da EspécieRESUMO
The therapeutic scope of antibody and nonantibody protein scaffolds is still prohibitively limited against intracellular drug targets. Here, we demonstrate that the Alphabody scaffold can be engineered into a cell-penetrating protein antagonist against induced myeloid leukemia cell differentiation protein MCL-1, an intracellular target in cancer, by grafting the critical B-cell lymphoma 2 homology 3 helix of MCL-1 onto the Alphabody and tagging the scaffold's termini with designed cell-penetration polypeptides. Introduction of an albumin-binding moiety extended the serum half-life of the engineered Alphabody to therapeutically relevant levels, and administration thereof in mouse tumor xenografts based on myeloma cell lines reduced tumor burden. Crystal structures of such a designed Alphabody in complex with MCL-1 and serum albumin provided the structural blueprint of the applied design principles. Collectively, we provide proof of concept for the use of Alphabodies against intracellular disease mediators, which, to date, have remained in the realm of small-molecule therapeutics.
Assuntos
Neoplasias , Peptídeos , Animais , Apoptose , Linhagem Celular , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Humanos , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Peptídeos/químicaRESUMO
In this study we constructed two phage libraries displaying non-immunized natural human IgM derived HCDR3 repertoires. One library was structurally constrained by a Gly to Cys substitution at position 104 enabling the formation of a disulfide bridge with the Cys at position 92. Panning of these libraries on an anti-human influenza hemagglutinin (HA) antibody resulted in the selection of 16 different HCDR3 loops displaying different degrees of sequence homology with the HA epitope. The specificity of the HCDR3 loops recovered from the structurally constrained library was confirmed by competition assays using the HA epitope. Only one of these HCDR3 peptides contained Cys104. Structural analysis of these sequences revealed that the loss of Cys104 was associated with an increased preference for the formation of the type I beta-turn required for high affinity binding to the antibody. Affinity studies confirmed that the HCDR3 peptides containing the sequence YDVPDY and Gly104 had affinities in the nanomolar range (K(d)=7.6 nM) comparable to the HA epitope. These findings provided evidence that the recovered HCDR3 sequences may bind to their target in a conformation that is unreachable by the parental antibody from which the HCDR3 was derived. Furthermore, the isolation of target-specific and high affinity binders demonstrates the value of HCDR3 libraries as a source of 'biologically randomized' sequences of human origin for the identification of peptidic lead molecules.
Assuntos
Regiões Determinantes de Complementaridade , Epitopos , Hemaglutininas/imunologia , Cadeias Pesadas de Imunoglobulinas , Influenza Humana/imunologia , Mimetismo Molecular , Peptídeos/imunologia , Sequência de Aminoácidos , Anticorpos , Afinidade de Anticorpos , Humanos , Imunoglobulina M , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/isolamento & purificação , Estrutura Secundária de ProteínaRESUMO
Methodologies to conjugate proteins to property-enhancing entities are highly sought after. We report a remarkably simple strategy for conjugating proteins bearing accessible cysteines to unprotected peptides containing a Cys(Scm) protecting group, which is introduced on-resin via a Cys(Acm) building block. The peptides employed for this proof of principle study are highly varied and structurally diverse, and undergo multiple on-resin decoration steps prior to conjugation. The methodology was applied to three different proteins, and proved to be efficient and site-selective. This twist on protecting group chemistry has led to a novel and generally applicable strategy for crossed-disulfide formation between proteins and peptides.
Assuntos
Ácido Fólico/química , Peptídeos/metabolismo , Proteínas/metabolismo , Western Blotting , Cisteína/química , Eletroforese em Gel de Poliacrilamida , Estrutura Molecular , Oxirredução , Peptídeos/química , Proteínas/químicaRESUMO
The interplay between aromatic stacking and hydrogen bonding in nucleobases has been investigated via high-level quantum chemical calculations. The experimentally observed stacking arrangement between consecutive bases in DNA and RNA/DNA double helices is shown to enhance their hydrogen bonding ability as opposed to gas phase optimized complexes. This phenomenon results from more repulsive electrostatic interactions as is demonstrated in a model system of cytosine stacked offset-parallel with substituted benzenes. Therefore, the H-bonding capacity of the N3 and O2 atoms of cytosine increases linearly with the electrostatic repulsion between the stacked rings. The local hardness, a density functional theory-based reactivity descriptor, appears to be a key index associated with the molecular electrostatic potential (MEP) minima around H-bond accepting atoms, and is inversely proportional to the electrostatic interaction between stacked molecules. Finally, the MEP minima on surfaces around the bases in experimental structures of DNA and RNA-DNA double helices show that their hydrogen bonding capacity increases when taking more neighboring (intra-strand) stacking partners into account.
Assuntos
DNA/química , RNA/química , Pareamento de Bases , Derivados de Benzeno/química , Biologia Computacional , Citosina/química , Ligação de Hidrogênio , Modelos Químicos , Estrutura Molecular , Eletricidade EstáticaRESUMO
It is well documented that helices in proteins can decrease the pKa of residues located at the N-terminus, but the real nature of this perturbation remains unclear. In the present work, the origin of the effect of 3(10)- and alpha-polyalanine helices on the pKa of an N-terminal cysteine residue is examined in gas phase as well as in aqueous solution by means of density functional theory. In a systematic study of the helix dipole, the proton affinity (PA), and the pKa of the N-terminal cysteine, in relation to both the helix length and the strength of the hydrogen bonds between the helix backbone amides and the Sgamma of the N-terminal cysteine, a direct relation between the terminal hydrogen bonds and the pKa perturbation is revealed.
Assuntos
Cisteína/química , Modelos Químicos , Peptídeos/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Estrutura Secundária de ProteínaRESUMO
General acid catalysis is a powerful and widely used strategy in enzymatic nucleophilic displacement reactions. For example, hydrolysis/phosphorolysis of the N-glycosidic bond in nucleosides and nucleotides commonly involves the protonation of the leaving nucleobase concomitant with nucleophilic attack. However, in the nucleoside hydrolase of the parasite Trypanosoma vivax, crystallographic and mutagenesis studies failed to identify a general acid. This enzyme binds the purine base of the substrate between the aromatic side-chains of Trp83 and Trp260. Here, we show via quantum chemical calculations that face-to-face stacking can raise the pKa of a heterocyclic aromatic compound by several units. Site-directed mutagenesis combined with substrate engineering demonstrates that Trp260 catalyzes the cleavage of the glycosidic bond by promoting the protonation of the purine base at N-7, hence functioning as an alternative to general acid catalysis.
Assuntos
N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismo , Trypanosoma vivax/enzimologia , Triptofano/metabolismo , Ácidos , Animais , Sítios de Ligação , Catálise , Cristalografia por Raios X , Ativação Enzimática , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Relação Estrutura-Atividade , Triptofano/químicaRESUMO
Ribonucleases (RNases) have proven to be excellent model systems for the study of protein structure, folding and stability, and enzyme catalysis, resulting in four Nobel Prize lectures in chemistry. Beside this 'academic' success, RNases are also relevant from a medical point of view. The RNA population in cells is controlled post-transcriptionally by ribonucleases (RNases) of varying specificity. Other therapeutic proteins like angiogenin, neurotoxins, and plant allergens have RNase activity or significant structural homology to known RNases. Also, RNase activity in serum and cell extracts is elevated in a variety of cancers and infectious diseases. To date, no clinical drugs are available that target this important class of enzymes. Small-molecule RNase inhibitors derived from mono- or dinucleotides, as well as pentavalent oxyvanadate transition state analogs are found to be rather marginal inhibitors. These compounds bind their target RNase with dissociation constants in the micromolar range, whereas transition state theory predicts picomolar values for genuine transition states. The rational design for new transition state analog inhibitors requires knowledge of the precise nature of the transition state and of the occurring intermolecular enzyme-substrate interactions. This review focuses on these chemical and structural features of RNase A and RNase T(1), the best characterized members of two separate classes of ribonucleases.
Assuntos
Ribonucleases/química , Animais , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Inibidores Enzimáticos/química , Humanos , Oxigênio/química , Ribonucleases/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
Protein scaffolds can provide a promising alternative to antibodies for various biomedical and biotechnological applications, including therapeutics. Here we describe the design and development of the Alphabody, a protein scaffold featuring a single-chain antiparallel triple-helix coiled-coil fold. We report affinity-matured Alphabodies with favourable physicochemical properties that can specifically neutralize human interleukin (IL)-23, a pivotal therapeutic target in autoimmune inflammatory diseases such as psoriasis and multiple sclerosis. The crystal structure of human IL-23 in complex with an affinity-matured Alphabody reveals how the variable interhelical groove of the scaffold uniquely targets a large epitope on the p19 subunit of IL-23 to harness fully the hydrophobic and hydrogen-bonding potential of tryptophan and tyrosine residues contributed by p19 and the Alphabody, respectively. Thus, Alphabodies are suitable for targeting protein-protein interfaces of therapeutic importance and can be tailored to interrogate desired design and binding-mode principles via efficient selection and affinity-maturation strategies.
Assuntos
Interleucina-23/antagonistas & inibidores , Peptídeos/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peptídeos/uso terapêutico , Psoríase/prevenção & controleRESUMO
Native mass spectrometry (MS) is a powerful technique for studying noncovalent protein-protein interactions. Here, native MS was employed to examine the noncovalent interactions involved in homodimerization of antibody half molecules (HL) in hinge-deleted human IgG4 (IgG4Δhinge). By analyzing the concentration dependence of the relative distribution of monomer HL and dimer (HL)(2) species, the apparent dissociation constant (K(D)) for this interaction was determined. In combination with site-directed mutagenesis, the relative contributions of residues at the CH3-CH3 interface to this interaction could be characterized and corresponding K(D) values quantified over a range of 10(-10)-10(-4) M. The critical importance of this noncovalent interaction in maintaining the intact dimeric structure was also proven for the full-length IgG4 backbone. Using time-resolved MS, the kinetics of the interaction could be measured, reflecting the dynamics of IgG4 HL exchange. Hence, native MS has provided a quantitative view of local structural features that define biological properties of IgG4.
Assuntos
Sítios de Ligação de Anticorpos , Fragmentos de Imunoglobulinas/química , Imunoglobulina G/química , Espectrometria de Massas por Ionização por Electrospray , Motivos de Aminoácidos , Simulação por Computador , Humanos , Fragmentos de Imunoglobulinas/genética , Imunoglobulina G/genética , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Ligação Proteica , Estabilidade Proteica , TermodinâmicaRESUMO
The reduction of arsenate to arsenite by pI258 arsenate reductase (ArsC) combines a nucleophilic displacement reaction with a unique intramolecular disulfide cascade. Within this reaction mechanism, the oxidative equivalents are translocated from the active site to the surface of ArsC. The first reaction step in the reduction of arsenate by pI258 ArsC consists of a nucleophilic displacement reaction carried out by Cys10 on dianionic arsenate. The second step involves the nucleophilic attack of Cys82 on the Cys10-arseno intermediate formed during the first reaction step. The onset of the second step is studied here by using quantum chemical calculations in a density functional theory context. The optimised geometry of the Cys10-arseno adduct in the ArsC catalytic site (sequence motif: Cys10-Thr11-Gly12-Asn13-Ser14-Cys15-Arg16-Ser17) forms the starting point for all subsequent calculations. Thermodynamic data and a hard and soft acids and bases (HSAB) reactivity analysis show a preferential nucleophilic attack on a monoanionic Cys10-arseno adduct, which is stabilised by Ser17. The P-loop active site of pI258 ArsC activates first a hydroxy group and subsequently arsenite as the leaving group, as is clear from an increase in the calculated nucleofugality of these groups upon going from the gas phase to the solvent phase to the enzymatic environment. Furthermore, the enzymatic environment stabilises the thiolate form of the nucleophile Cys82 by 3.3 pH units through the presence of the eight-residue alpha helix flanked by Cys82 and Cys89 (redox helix) and through a hydrogen bond with Thr11. The importance of Thr11 in the pKa regulation of Cys82 was confirmed by the observed decrease in the kcat value of the Thr11Ala mutant as compared to that of wild-type ArsC. During the final reaction step, Cys89 is activated as a nucleophile by structural alterations of the redox helix that functions as a pKa control switch for Cys89; this final step is necessary to expose a Cys82-Cys89 disulfide.
Assuntos
Arseniatos/química , Bombas de Íon/química , Complexos Multienzimáticos/química , Staphylococcus aureus/enzimologia , ATPases Transportadoras de Arsenito , Catálise , Simulação por Computador , Cisteína/química , Eletroquímica , Bombas de Íon/isolamento & purificação , Modelos Moleculares , Estrutura Molecular , Complexos Multienzimáticos/isolamento & purificação , MutaçãoRESUMO
In enzymatic depurination of nucleosides, the 5'-OH group of the ribose moiety of the substrate is often shown to contribute substantially to catalysis. The purine-specific nucleoside hydrolase from Trypanosoma vivax (TvNH) fixes the 5'-OH group in a gauche,trans orientation about the C4'-C5' bond, enabling the 5'-oxygen to accept an intramolecular hydrogen bond from the C8-atom of the purine leaving group. High level ab initio quantum chemical calculations indicate that this interaction promotes protonation of the purine at N7. Steady state kinetics comprising engineered substrates confirm that a considerable fraction of the catalytic 5'-OH effect can be attributed to leaving group activation.
Assuntos
Glicosídeos/química , Trypanosoma vivax/enzimologia , Animais , Catálise , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Íons , Cinética , Modelos Químicos , Modelos Moleculares , Mutagênese , Oxigênio/química , Ligação Proteica , Conformação Proteica , Purinas/química , Especificidade por Substrato , Triptofano/químicaRESUMO
Ribonucleases (RNases) catalyze the cleavage of the phosphodiester bond in RNA up to 10(15)-fold, as compared with the uncatalyzed reaction. High resolution crystal structures of these enzymes in complex with 3'-mononucleotide substrates demonstrate the accommodation of the nucleophilic 2'-OH group in a binding pocket comprising the catalytic base (glutamate or histidine) and a charged hydrogen bond donor (lysine or histidine). Ab initio quantum chemical calculations performed on such Michaelis complexes of the mammalian RNase A (EC ) and the microbial RNase T(1) (EC ) show negative charge build up on the 2'-oxygen upon substrate binding. The increased nucleophilicity results from stronger hydrogen bonding to the catalytic base, which is mediated by a hydrogen bond from the charged donor. This hitherto unrecognized catalytic dyad in ribonucleases constitutes a general mechanism for nucleophile activation in both enzymic and RNA-catalyzed phosphoryl transfer reactions.