Nanoscale spatial dependence of avidity in an IgG1 antibody.

Antibodies are secreted proteins that are crucial to recognition of pathogens by the immune system and are also efficient pharmaceuticals. The affinity and specificity of target recognition can increase remarkably through avidity effects, when the antibody can bind a multivalent antigen through more than one epitope simultaneously. A key goal of antibody engineering is thus to optimize avidity, but little is known about the nanoscale spatial dependence of avidity in antibodies. Here, we develop a set of anti-parallel coiled-coils spanning from 7 to 20 nm and validate their structure using biophysical techniques. We use the coiled-coils to control the spacing between two epitopes, and measure how antigen spacing affects the stability of the bivalent antibody:antigen complex. We find a maximal avidity enhancement at a spacing of 13 nm. In contrast to recent studies, we find the avidity to be relatively insensitive to epitope spacing near the avidity maximum as long as it is within the spatial tolerance of the antibody. We thus only see a ~ twofold variation of avidity in the range from 7 to 20 nm. The coiled-coil systems developed here may prove a useful protein nanocaliper for profiling the spatial tolerance and avidity profile of bispecific antibodies.

Linker Dependence of Avidity in Multivalent Interactions Between Disordered Proteins.

Multidomain proteins often interact through several independent binding sites connected by disordered linkers. The architecture of such linkers affects avidity by modulating the effective concentration of intramolecular binding. The linker dependence of avidity has been estimated theoretically using simple physical models, but such models have not been tested experimentally because the effective concentrations could not be measured directly. We have developed a model system for bivalent protein interactions connected by disordered linkers, where the effective concentration can be measured using a competition experiment. We characterized the bivalent protein interactions kinetically and thermodynamically for a variety of linker lengths and interaction strengths. In total, this allowed us to critically assess the existing theoretical models of avidity in disordered, multivalent interactions. As expected, the onset of avidity occurs when the effective concentration reached the dissociation constant of the weakest interaction. Avidity decreased monotonously with linker length, but only by a third of what is predicted by theoretical models. We suggest that the length dependence of avidity is attenuated by compensating mechanisms such as linker interactions or entanglement. The direct role of linkers in avidity suggests they provide a generic mechanism for allosteric regulation of disordered, multivalent proteins.

Production of Lysostaphin by Nonproprietary Method Utilizing a Promoter from Toxin-Antitoxin System.

Lysostaphin is a staphylolytic protein of growing interest from biotechnological and pharmaceutical industry due to its potential use in preventing and combating staphylococcal infections. Here, we describe an optimized method for production of lysostaphin in an inductionless system utilizing constitutive promoter from staphylococcal toxin-antitoxin system PemIK-Sa1. We investigated the influence of ribosome-binding site sequence, Escherichia coli producer strain and growth media on yield and kinetics of recombinant protein production. Lysostaphin was purified in its native active form using one-step cation-exchange chromatography. The system provides a method for cost-efficient and scalable protein production, and can be applied to produce other biotechnologically significant proteins.

Biochemical and structural analyses suggest that plasminogen activators coevolved with their cognate protein substrates and inhibitors.

Protein sequences of members of the plasminogen activation system are present throughout the entire vertebrate phylum. This important and well-described proteolytic cascade is governed by numerous protease-substrate and protease-inhibitor interactions whose conservation is crucial to maintaining unchanged protein function throughout evolution. The pressure to preserve protein-protein interactions may lead to either co-conservation or covariation of binding interfaces. Here, we combined covariation analysis and structure-based prediction to analyze the binding interfaces of urokinase (uPA):plasminogen activator inhibitor-1 (PAI-1) and uPA:plasminogen complexes. We detected correlated variation between the S3-pocket-lining residues of uPA and the P3 residue of both PAI-1 and plasminogen. These residues are known to form numerous polar interactions in the human uPA:PAI-1 Michaelis complex. To test the effect of mutations that correlate with each other and have occurred during mammalian diversification on protein-protein interactions, we produced uPA, PAI-1, and plasminogen from human and zebrafish to represent mammalian and nonmammalian orthologs. Using single amino acid point substitutions in these proteins, we found that the binding interfaces of uPA:plasminogen and uPA:PAI-1 may have coevolved to maintain tight interactions. Moreover, we conclude that although the interaction areas between protease-substrate and protease-inhibitor are shared, the two interactions are mechanistically different. Compared with a protease cleaving its natural substrate, the interaction between a protease and its inhibitor is more complex and involves a more fine-tuned mechanism. Understanding the effects of evolution on specific protein interactions may help further pharmacological interventions of the plasminogen activation system and other proteolytic systems.

Origin and diversification of the plasminogen activation system among chordates.

BACKGROUND: The plasminogen (PLG) activation system is composed by a series of serine proteases, inhibitors and several binding proteins, which together control the temporal and spatial generation of the active serine protease plasmin. As this proteolytic system plays a central role in human physiology and pathophysiology it has been extensively studied in mammals. The serine proteases of this system are believed to originate from an ancestral gene by gene duplications followed by domain gains and deletions. However, the identification of ancestral forms in primitive chordates supporting these theories remains elusive. In addition, evolutionary studies of the non-proteolytic members of this system are scarce. RESULTS: Our phylogenetic analyses place lamprey PLG at the root of the vertebrate PLG-group, while lamprey PLG-related growth factors represent the ancestral forms of the jawed-vertebrate orthologues. Furthermore, we find that the earliest putative orthologue of the PLG activator group is the hyaluronan binding protein 2 (HABP2) gene found in lampreys. The prime plasminogen activators (tissue- and urokinase-type plasminogen activator, tPA and uPA) first occur in cartilaginous fish and phylogenetic analyses confirm that all orthologues identified compose monophyletic groups to their mammalian counterparts. Cartilaginous fishes exhibit the most ancient vitronectin of all vertebrates, while plasminogen activator inhibitor 1 (PAI-1) appears for the first time in cartilaginous fishes and is conserved in the rest of jawed vertebrate clades. PAI-2 appears for the first time in the common ancestor of reptiles and mammals, and represents the latest appearing plasminogen activator inhibitor. Finally, we noted that the urokinase-type plasminogen activator receptor (uPAR)-and three-LU domain containing genes in general-occurred later in evolution and was first detectable after coelacanths. CONCLUSIONS: This study identifies several primitive orthologues of the mammalian plasminogen activation system. These ancestral forms provide clues to the origin and diversification of this enzyme system. Further, the discovery of several members-hitherto unknown in mammals-provide new perspectives on the evolution of this important enzyme system.

Allosteric mechanisms underlying the adaptive increase in hemoglobin-oxygen affinity of the bar-headed goose.

The high blood-O2 affinity of the bar-headed goose (Anser indicus) is an integral component of the biochemical and physiological adaptations that allow this hypoxia-tolerant species to undertake migratory flights over the Himalayas. The high blood-O2 affinity of this species was originally attributed to a single amino acid substitution of the major hemoglobin (Hb) isoform, HbA, which was thought to destabilize the low-affinity T state, thereby shifting the T-R allosteric equilibrium towards the high-affinity R state. Surprisingly, this mechanistic hypothesis has never been addressed using native proteins purified from blood. Here, we report a detailed analysis of O2 equilibria and kinetics of native major HbA and minor HbD isoforms from bar-headed goose and greylag goose (Anser anser), a strictly lowland species, to identify and characterize the mechanistic basis for the adaptive change in Hb function. We find that HbA and HbD of bar-headed goose have consistently higher O2 affinities than those of the greylag goose. The corresponding Hb isoforms of the two species are equally responsive to physiological allosteric cofactors and have similar Bohr effects. Thermodynamic analyses of O2 equilibrium curves according to the two-state Monod-Wyman-Changeaux model revealed higher R-state O2 affinities in the bar-headed goose Hbs, associated with lower O2 dissociation rates, compared with the greylag goose. Conversely, the T state was not destabilized and the T-R allosteric equilibrium was unaltered in bar-headed goose Hbs. The physiological implication of these results is that increased R-state affinity allows for enhanced O2 saturation in the lungs during hypoxia, but without impairing O2 delivery to tissues.

Molecular basis of hemoglobin adaptation in the high-flying bar-headed goose.

During the adaptive evolution of a particular trait, some selectively fixed mutations may be directly causative and others may be purely compensatory. The relative contribution of these two classes of mutation to adaptive phenotypic evolution depends on the form and prevalence of mutational pleiotropy. To investigate the nature of adaptive substitutions and their pleiotropic effects, we used a protein engineering approach to characterize the molecular basis of hemoglobin (Hb) adaptation in the high-flying bar-headed goose (Anser indicus), a hypoxia-tolerant species renowned for its trans-Himalayan migratory flights. To test the effects of observed substitutions on evolutionarily relevant genetic backgrounds, we synthesized all possible genotypic intermediates in the line of descent connecting the wildtype bar-headed goose genotype with the most recent common ancestor of bar-headed goose and its lowland relatives. Site-directed mutagenesis experiments revealed one major-effect mutation that significantly increased Hb-O2 affinity on all possible genetic backgrounds. Two other mutations exhibited smaller average effect sizes and less additivity across backgrounds. One of the latter mutations produced a concomitant increase in the autoxidation rate, a deleterious side-effect that was fully compensated by a second-site mutation at a spatially proximal residue. The experiments revealed three key insights: (i) subtle, localized structural changes can produce large functional effects; (ii) relative effect sizes of function-altering mutations may depend on the sequential order in which they occur; and (iii) compensation of deleterious pleiotropic effects may play an important role in the adaptive evolution of protein function.

Functional diversification of sea lamprey globins in evolution and development.

Agnathans have a globin repertoire that markedly differs from that of jawed (gnathostome) vertebrates. The sea lamprey (Petromyzon marinus) harbors at least 18 hemoglobin, two myoglobin, two globin X, and one cytoglobin genes. However, agnathan hemoglobins and myoglobins are not orthologous to their cognates in jawed vertebrates. Thus, blood-based O2 transport and muscle-based O2 storage proteins emerged twice in vertebrates from a tissue-globin ancestor. Notably, the sea lamprey displays three switches in hemoglobin expression in its life cycle, analogous to hemoglobin switching in vertebrates. To study the functional changes associated with the evolution and ontogenesis of distinct globin types, we determined O2 binding equilibria, type of quaternary assembly, and nitrite reductase enzymatic activities of one adult (aHb5a) and one embryonic/larval hemoglobin (aHb6), myoglobin (aMb1) and cytoglobin (Cygb) of the sea lamprey. We found clear functional differentiation among globin types expressed at different developmental stages and in different tissues. Cygb and aMb1 have high O2 affinity and nitrite reductase activity, while the two hemoglobins display low O2 affinity and nitrite reductase activity. Cygb and aHb6 but not aHb5a show cooperative O2 binding, correlating with increased stability of dimers, as shown by gel filtration and molecular modeling. The high O2-affinity and the lack of cooperativity confirm the identity of the sea lamprey aMb1 as O2 storage protein of the muscle. The dimeric structure and O2-binding properties of sea lamprey and mammalian Cygb were very similar, suggesting a conservation of function since their divergence around 500million years ago.

Latency transition of plasminogen activator inhibitor type 1 is evolutionarily conserved.

Plasminogen activator inhibitor type 1 (PAI-1) is a central regulator of fibrinolysis and tissue remodelling. PAI-1 belongs to the serpin superfamily and unlike other inhibitory serpins undergoes a spontaneous inactivation process under physiological conditions, termed latency transition. During latency transition the solvent exposed reactive centre loop is inserted into the central ß-sheet A of the molecule, and is no longer accessible to reaction with the protease. More than three decades of research on mammalian PAI-1 has not been able to clarify the evolutionary advantage and physiological relevance of latency transition. In order to study the origin of PAI-1 latency transition, we produced PAI-1 from Spiny dogfish shark (Squalus acanthias) and African lungfish (Protopterus sp.), which represent central species in the evolution of vertebrates. Although human PAI-1 and the non-mammalian PAI-1 variants share only approximately 50 % sequence identity, our results showed that all tested PAI-1 variants undergo latency transition with a similar rate. Since the functional stability of PAI-1 can be greatly increased by substitution of few amino acid residues, we conclude that the ability to undergo latency transition must have been a specific selection criterion for the evolution of PAI-1. It appears that all PAI-1 molecules must harbour latency transition to fulfil their physiological function, stressing the importance to further pursue a complete understanding of this molecular phenomenon with possible implication to pharmacological intervention. Our results provide the next step in understanding how the complete role of this important protease inhibitor evolved along with the fibrinolytic system.

Multi-tissue RNA-seq and transcriptome characterisation of the spiny dogfish shark (Squalus acanthias) provides a molecular tool for biological research and reveals new genes involved in osmoregulation.

The spiny dogfish shark (Squalus acanthias) is one of the most commonly used cartilaginous fishes in biological research, especially in the fields of nitrogen metabolism, ion transporters and osmoregulation. Nonetheless, transcriptomic data for this organism is scarce. In the present study, a multi-tissue RNA-seq experiment and de novo transcriptome assembly was performed in four different spiny dogfish tissues (brain, liver, kidney and ovary), providing an annotated sequence resource. The characterization of the transcriptome greatly increases the scarce sequence information for shark species. Reads were assembled with the Trinity de novo assembler both within each tissue and across all tissues combined resulting in 362,690 transcripts in the combined assembly which represent 289,515 Trinity genes. BUSCO analysis determined a level of 87% completeness for the combined transcriptome. In total, 123,110 proteins were predicted of which 78,679 and 83,164 had significant hits against the SwissProt and Uniref90 protein databases, respectively. Additionally, 61,215 proteins aligned to known protein domains, 7,208 carried a signal peptide and 15,971 possessed at least one transmembrane region. Based on the annotation, 81,582 transcripts were assigned to gene ontology terms and 42,078 belong to known clusters of orthologous groups (eggNOG). To demonstrate the value of our molecular resource, we show that the improved transcriptome data enhances the current possibilities of osmoregulation research in spiny dogfish by utilizing the novel gene and protein annotations to investigate a set of genes involved in urea synthesis and urea, ammonia and water transport, all of them crucial in osmoregulation. We describe the presence of different gene copies and isoforms of key enzymes involved in this process, including arginases and transporters of urea and ammonia, for which sequence information is currently absent in the databases for this model species. The transcriptome assemblies and the derived annotations generated in this study will support the ongoing research for this particular animal model and provides a new molecular tool to assist biological research in cartilaginous fishes.

Protein conformational change delayed by steric hindrance from an N-linked glycan.

Very few studies have attributed a direct, active, functional role to N-linked glycans. We describe here an N-linked glycan with a unique role for maintaining the active conformation of a protein of the serpin family. The distinguishing feature of serpins is the "stressed-to-relaxed" transition, in which the reactive center loop inserts as a ß-strand into the central ß-sheet A. This transition forms the basis for the conversion of serpins to the inactive latent state. We demonstrate that plasminogen activator inhibitor-1 (PAI-1) from zebrafish converts to the latent state about 5-fold slower than human PAI-1. In contrast to human PAI-1, fish PAI-1 carries a single N-linked glycan at Asn185 in the gate region through which the reactive center loop passes during latency transition. While the latency transition of human PAI-1 is unaffected by deglycosylation, deglycosylated zebrafish PAI-1 (zfPAI-1) goes latent about 50-fold faster than the glycosylated zfPAI-1 and about 25-fold faster than non-glycosylated human PAI-1. X-ray crystal structure analysis of glycosylated fish PAI-1 confirmed the presence of an N-linked glycan in the gate region and a lack of glycan-induced structural changes. Thus, latency transition of zfPAI-1 is delayed by steric hindrance from the glycan in the gate region. Our findings reveal a previously unknown mechanism for inhibition of protein conformational changes by steric hindrance from N-linked glycans.