Comparing domain interactions within antibody Fabs with kappa and lambda light chains

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.     

Relative stabilities of IgG1 and IgG4 Fab domains: Influence of the light-heavy interchain disulfide bond architecture

The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 C(H) 1 and IgG4 C(H) 1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L-H) bond, did not affect thermal stability. Introducing the IgG1 type L-H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L-H interchain DSB with the IgG4 type L-H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 C(H) 1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format.     

Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies.     

Metal ions and phosphate binding in the H-N-H motif: crystal structures of the nuclease domain of ColE7/Im7 in complex with a phosphate ion and different divalent metal ions

H-N-H is a motif found in the nuclease domain of a subfamily of bacteria toxins, including colicin E7, that are capable of cleaving DNA nonspecifically. This H-N-H motif has also been identified in a subfamily of homing endonucleases, which cleave DNA site specifically. To better understand the role of metal ions in the H-N-H motif during DNA hydrolysis, we crystallized the nuclease domain of colicin E7 (nuclease-ColE7) in complex with its inhibitor Im7 in two different crystal forms, and we resolved the structures of EDTA-treated, Zn(2+)-bound and Mn(2+)-bound complexes in the presence of phosphate ions at resolutions of 2.6 A to 2.0 A. This study offers the first determination of the structure of a metal-free and substrate-free enzyme in the H-N-H family. The H-N-H motif contains two antiparallel beta-strands linked to a C-terminal alpha-helix, with a divalent metal ion located in the center. Here we show that the metal-binding sites in the center of the H-N-H motif, for the EDTA-treated and Mg(2+)-soaked complex crystals, were occupied by water molecules, indicating that an alkaline earth metal ion does not reside in the same position as a transition metal ion in the H-N-H motif. However, a Zn(2+) or Mn(2+) ions were observed in the center of the H-N-H motif in cases of Zn(2+) or Mn(2+)-soaked crystals, as confirmed in anomalous difference maps. A phosphate ion was found to bridge between the divalent transition metal ion and His545. Based on these structures and structural comparisons with other nucleases, we suggest a functional role for the divalent transition metal ion in the H-N-H motif in stabilizing the phosphoanion in the transition state during hydrolysis.     

Recombinant enterokinase light chain with affinity tag: expression from Saccharomyces cerevisiae and its utilities in fusion protein technology.

Enterokinase and recombinant enterokinase light chain (rEK(L)) have been used widely to cleave fusion proteins with the target sequence of (Asp)(4)-Lys. In this work, we show that their utility as a site-specific cleavage agent is compromised by sporadic cleavage at other sites, albeit at low levels. Further degradation of the fusion protein in cleavage reaction is due to an intrinsic broad specificity of the enzyme rather than to the presence of contaminating proteases. To offer facilitated purification from fermentation broth and efficient removal of rEK(L) after cleavage reaction, thus minimizing unwanted cleavage of target protein, histidine affinity tag was introduced into rEK(L). Utilizing the secretion enhancer peptide derived from the human interleukin 1 beta, the recombinant EK(L) was expressed in Saccharomyces cerevisiae and efficiently secreted into culture medium. The C-terminal His-tagged EK(L) was purified in a single-step procedure on nickel affinity chromatography. It retained full enzymatic activity similar to that of EK(L), whereas the N-terminal His-tagged EK(L) was neither efficiently purified nor had any enzymatic activity. After cleavage reaction of fusion protein, the C-terminal His-tagged EK(L) was efficiently removed from the reaction mixture by a single passage through nickel-NTA spin column. The simple affinity tag renders rEK(L) extremely useful for purification, post-cleavage removal, recovery, and recycling and will broaden the utility and the versatility of the enterokinase for the production of recombinant proteins.     

金属离子对漆斑菌产胆红素氧化酶的影响

比较了漆斑菌在8种液体培养基中胆红素氧化酶(BOX)产量,发现马铃薯液体培养基(PDB)是最适宜漆斑菌产BOX的培养基。研究了8种常见金属离子对漆斑菌产酶的影响,结果表明钠离子、铜离子可以明显促进BOX产量提高,铜离子效果最强,随着铜离子浓度增加,BOX酶产量可进一步提高,但高浓度的铜离子(1mmol.L-1)会抑制酶产量增加。     

人心肌肌球蛋白轻链1与重链和肌动蛋白的结合

在测得中国人心肌肌球蛋白轻链 1cDNA的核苷酸序列 ,并获得一株单克隆抗体 (HCMLC1 8)的基础上 ,用PCR方法 ,以中国人心肌肌球蛋白轻链 1的cDNA为模板 ,分别获得中国人心肌肌球蛋白轻链 1的各为 98个氨基酸的N端和C端片段cDNA的克隆并进行了表达。同时进行了其表达产物和大鼠心肌肌球蛋白重链和人心肌肌动蛋白以及单克隆抗体结合的研究 ,发现三者均和轻链 1的N端相结合 ,结合位点各不相同。这些结合位点可能均位于轻链 1的分子表面 ,而且如果轻链 1在实验状态下先与肌动蛋白结合 ,则有可能影响轻链与重链间的彼此结合。肌动蛋白在体外能以不同位点结合肌球蛋白重链和轻链 ,可能在肌肉收缩过程中具有重要的生理意义     

二价金属离子转运蛋白1——一个新发现的重要铁转运蛋白

二价金属离子转运蛋白1(divalent metal transporter 1,DMT1)的发现是近年铁代谢研究领域最重大的一项突破.DMT1是哺乳类跨膜铁转运蛋白.这种蛋白质广泛分布于人体各组织.DMT1 mRNA有两种形式,一种含有IRE(iron response element),而另一种则不含此结构.DMT1的功能主要是介导小肠上皮细胞的铁吸收以及参与铁从内吞小体移位到胞浆的过程.DMT1介导的铁转运是一个主动的和H⁺依赖的过程.DMT1也参与其他二价金属如Zn²⁺、Mn²⁺、Co²⁺、Cd²⁺、Cn²⁺、Ni²⁺和Pb²⁺的转运.小肠DMT1的表达受饮食或组织铁控制.第四跨膜区是DMT1的重要功能区.此区基因发生点突变（G185R）是导致不可逆性缺铁性贫血的原因.在帕金森氏病人的黑质发现DMT1表达异常增加,因而DMT1可能也与某些神经退行性疾病的形成有关.     

Cross-reactivity studies of an anti-Plasmodium vivax apical membrane antigen 1 monoclonal antibody: binding and structural characterisation

Apical membrane antigen 1 (AMA1) has an important, but as yet uncharacterised, role in host cell invasion by the malaria parasite, Plasmodium. The protein, which is quite conserved between Plasmodium species, comprises an ectoplasmic region, a single transmembrane segment and a small cytoplasmic domain. The ectoplasmic region, which can induce protective immunity in animal models of human malaria, is a leading vaccine candidate that has entered clinical trials. The monoclonal antibody F8.12.19, raised against the recombinant ectoplasmic region of AMA1 from Plasmodium vivax, cross-reacts with homologues from Plasmodium knowlesi, Plasmodium cynomolgi, Plasmodium berghei and Plasmodium falciparum, as shown by immunofluorescence assays on mature schizonts. The binding of F8.12.19 to recombinant AMA1 from both P. vivax and P. falciparum was measured by surface plasmon resonance, revealing an apparent affinity constant that is about 100-fold weaker for the cross-reacting antigen when compared to the cognate antigen. Crystal structure analysis of Fab F8.12.19 complexed to AMA1 from P. vivax and P. falciparum shows that the monoclonal antibody recognises a discontinuous epitope located on domain III of the ectoplasmic region, the major component being a loop containing a cystine knot. The structures provide a basis for understanding the cross-reactivity. Antibody contacts are made mainly to main-chain and invariant side-chain atoms of AMA1; contact antigen residues that differ in sequence are located at the periphery of the antigen-binding site and can be accommodated at the interface between the two components of the complex. The implications for AMA1 vaccine development are discussed.     

Janus kinase 2 modulates the lipid-removing but not protein-stabilizing interactions of amphipathic helices with ABCA1

ABCA1 mediates the transport of cellular cholesterol and phospholipids to HDL apolipoproteins. Apolipoprotein A-I (apoA-I) interactions with ABCA1-expressing cells elicit several responses, including removing cellular lipids, stabilizing ABCA1 protein, and activating Janus kinase 2 (JAK2). Here, we used synthetic apolipoprotein-mimetic peptides to characterize the relationship between these responses. Peptides containing one amphipathic helix of L- or D-amino acids (2F, D-2F, or 4F) and a peptide containing two helices (37pA) all promoted ABCA1-dependent cholesterol efflux, competed for apoA-I binding to ABCA1-expressing cells, blocked covalent cross-linking of apoA-I to ABCA1, and inhibited ABCA1 degradation. 37pA was cross-linked to ABCA1, confirming the direct binding of amphipathic helices to ABCA1. 2F, 4F, 37pA, and D-37pA all stimulated JAK2 autophosphorylation. Inhibition of JAK2 greatly reduced peptide-mediated cholesterol efflux, peptide binding to ABCA1-expressing cells, and peptide cross-linking to ABCA1, indicating that these processes require an active JAK2. In contrast, apoA-I and peptides stabilized ABCA1 protein even in the absence of an active JAK2, implying that this process is independent of JAK2 and lipid efflux-promoting binding of amphipathic helices to ABCA1. These findings show that amphipathic helices coordinate the activity of ABCA1 by several distinct mechanisms that are likely to involve different cell surface binding sites.     

Molecular cloning and expression analysis of a myosin light chain 1 (MLC‐1) gene from Indian meal moth Plodia interpunctella (Lepidoptera: Pyralidae)

Myosin light chain 1 (MLC‐1) protein acts in the organization, dynamics and transport processes associated with the cytoskeleton. In this work, an MLC‐1 gene was cloned and characterized from the Indian meal moth, Plodia interpunctella (Lepidoptera: Pyralidae). The isolated PiMLC‐1 cDNA is 913 bp, including a 5′‐untranslated region (UTR) of 79 bp, 3′‐UTR of 381 bp and an open reading frame (ORF) of 453 bp encoding a polypeptide of 150 amino acids, which contains two calcium binding domains (EF‐hands). The deduced PiMLC‐1 protein sequence has 39–94% comparison with other individuals. The qPCR analysis revealed that PiMLC‐1 was expressed in the four developmental stages (egg, larva, pupa and adult) and in all tissues tested, suggesting that it plays an important role in development of P. interpunctella. Based on the MLC‐1 amino acids, phylogenetic analysis showed a similar topology with the traditional classification, suggesting the potential value of the MLC‐1 protein in phylogenetic inference.     

Evaluation of preferred binding regions on ubiquitin and IgG1 FC for interacting with multimodal cation exchange resins using DEPC labeling/mass spectrometry

There is significant interest in identifying the preferred binding domains of biological products to various chromatographic materials. In this work, we develop a biophysical technique that uses diethyl pyrocarbonate (DEPC) based covalent labeling in concert with enzymatic digestion and mass spectrometry to identify the binding patches for proteins bound to commercially available multimodal (MM) cation exchange chromatography resins. The technique compares the changes in covalent labeling of the protein in solution and in the bound state and uses the differences in this labeling to identify residues that are sterically shielded upon resin binding and, therefore, potentially involved in the resin binding process. Importantly, this approach enables the labeling of many amino acids and can be carried out over a pH range of 5.5–7.5, thus enabling the protein surface mapping at conditions of interest in MM cation exchange systems. The protocol is first developed using the model protein ubiquitin and the results indicate that lysine residues located on the front face of the protein show dramatic changes in DEPC labeling while residues present on other regions have minimal or no reductions. This indicates that the front face of ubiquitin is likely involved in resin binding. In addition, surface property maps indicate that the hypothesized front face binding region consists of overlapping positively charged and hydrophobic patches. The technique is then employed with an IgG1 F_C and the results indicate that residues on the C_H2–C_H3 interface and the hinge are significantly sterically shielded upon binding to the resin. Further, these regions are again associated with significant overlap of positively charged and hydrophobic patches. On the other hand, while, residues on the C_H2 and the front face of the IgG1 F_C also exhibited some changes in DEPC labeling upon binding, these regions have less distinct charged and hydrophobic patches. Importantly, the hypothesized binding patches identified for both ubiquitin and F_C using this approach are shown to be consistent with previously reported NMR studies. In contrast to NMR, this new approach enables the identification of preferred binding regions without the need for isotopically labeled proteins or chemical shift assignments. The technique developed in this work sets the stage for the evaluation of the binding domains of a wide range of biological products to chromatographic surfaces, with important implications for designing biomolecules with improved biomanufacturability properties.     

Design of improved protein inhibitors of HIV-1 cell entry: Optimization of electrostatic interactions at the binding interface

Continuum electrostatic methods are a powerful tool for the analysis and design of biomolecular complexes, with methodologies that allow for the detailed analysis of the electrostatic contributions to binding affinities and procedures for computing the properties of electrostatically optimal ligands. We have applied these methods to the design of improved inhibitors of HIV-1 cell entry. HIV infection of a cell requires viral-cell membrane fusion, an event partially driven by a large-scale conformational change in the viral membrane glycoprotein gp41. This transformation involves the docking of a helix from the C-terminal region of three gp41 chains against a pre-formed trimeric-coiled coil; several protein constructs that inhibit membrane fusion act by binding to an isolated C-terminal helix and blocking the formation of the fusogenic structure. A detailed analysis of the electrostatic contributions to the binding of one such inhibitor (5-Helix) to a C-terminal helix was performed using the X-ray crystal structure of the core of the HIV-1 gp41 ectodomain as a structural model, and several residues on 5-Helix that make substantial contributions to binding, both favorable and unfavorable, were identified. An electrostatic affinity optimization methodology was applied to the side chains of 5-Helix, with the results showing that significant improvements in binding affinity are possible if the electrostatic contributions to the binding free energy are optimized. Several mutations accessible by experimental methods are suggested, with calculated improvements in binding affinity of as much as 500-fold and greater.     

Computational analysis of molecular basis of 1:1 interactions of NRG-1beta wild-type and variants with ErbB3 and ErbB4

The neuregulin/ErbB system is a growth factor/receptor cascade that has been proven to be essential in the development of the heart and the sympathetic nervous system. However, the basis of the specificity of ligand-receptor recognition remains to be elucidated. In this study, the structures of NRG-1beta/ErbB3 and NRG-1beta/ErbB4 complexes were modeled based on the available structures of the homologous proteins. The binding free energies of NRG-1beta to ErbB3 and ErbB4 were calculated using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) computational method. In addition, computational alanine-scanning mutagenesis was performed in the binding site of NRG-1beta and the difference in the binding free energies between NRG-1beta mutants and the receptors was calculated. The results specify the contribution of each residue at the interaction interfaces to the binding affinity of NRG-1beta with ErbB3 and ErbB4, identifying several important interaction residue pairs that are in agreement with previously acquired experimental data. This indicates that the presented structural models of NRG-1beta/ErbB3 and NRG-1beta/ErbB4 complexes are reliable and could be used to guide future studies, such as performing desirable mutations on NRG-1beta to increase the binding affinity and selectivity to the receptor and discovering new therapeutic agents for the treatment of heart failure.     

RACK1 (receptor for activated C‐kinase 1) interactions with spectrin repeat elements

Receptor for activated C‐kinase 1 (RACK1) is an intracellular scaffolding protein involved in a multitude of signalling pathways. The cytoskeleton is fundamental for intracellular cell signalling as it forms an interconnected network of regulatory proteins. Here, spectrin is a central component as it forms the actin–spectrin network that serves as docking surfaces for cellular components. The interaction between RACK1 and components of spectrin, the single spectrin repeats R16, R17 and the double spectrin repeat R1617 from the α‐spectrin chain were investigated by biosensor technology and docking analysis. RACK1 associated only weakly to R16 (K_D = 1.0 ± 0.5 × 10^?6 M), about 20 times stronger to R1617 (K_D = 5.3 ± 0.7 × 10^?8 M) and 100 times stronger to R17 (K_D = 0.9 ± 0.3 × 10^?8 M). Docking analysis showed that while R16 alone preferentially docked with its B‐helix, R17 docked through its A‐helix and BC loop. The double repeat and RACK1 mainly formed two different complex conformations. R1617 docked tangentially to the N/C‐terminal of RACK1 or radially along a groove on the outer surface of RACK1. These configurations could account for the slight increase in entropic and the decrease in enthalpic interactions for the R1617–RACK1 interaction, compared with the interactions of RACK1 to the two single repeats. Our results suggest a mode of interaction that allows spectrin to attach to the N/C part of RACK through the inter‐helical AB and BC loops and adopt a multitude of configurations in between the two limiting configurations. Copyright © 2014 John Wiley & Sons, Ltd.