Modification of the loops in the ligand-binding site turns avidin into a steroid-binding protein.

BACKGROUND: Engineered proteins, with non-immunoglobulin scaffolds, have become an important alternative to antibodies in many biotechnical and therapeutic applications. When compared to antibodies, tailored proteins may provide advantageous properties such as a smaller size or a more stable structure. RESULTS: Avidin is a widely used protein in biomedicine and biotechnology. To tailor the binding properties of avidin, we have designed a sequence-randomized avidin library with mutagenesis focused at the loop area of the binding site. Selection from the generated library led to the isolation of a steroid-binding avidin mutant (sbAvd-1) showing micromolar affinity towards testosterone (Kd ~ 9 µM). Furthermore, a gene library based on the sbAvd-1 gene was created by randomizing the loop area between ß-strands 3 and 4. Phage display selection from this library led to the isolation of a steroid-binding protein with significantly decreased biotin binding affinity compared to sbAvd-1. Importantly, differential scanning calorimetry and analytical gel-filtration revealed that the high stability and the tetrameric structure were preserved in these engineered avidins. CONCLUSIONS: The high stability and structural properties of avidin make it an attractive molecule for the engineering of novel receptors. This methodology may allow the use of avidin as a universal scaffold in the development of novel receptors for small molecules.

Chimeric avidin shows stability against harsh chemical conditions--biochemical analysis and 3D structure.

Avidin and its bacterial analog streptavidin have been widely used in applications in life sciences. Recently, we described a highly thermostable engineered avidin, called chimeric avidin, which is a hybrid of avidin and avidin-related protein 4. Here, we report a protocol for pilot-scale production in E. coli and the X-ray structure of chimeric avidin. The ligand-binding properties of chimeric avidin were explored with isothermal titration calorimetry. We found chimeric avidin to be more stable against various harsh organic solvents at elevated temperatures compared to avidin and streptavidin. The properties of chimeric avidin make it a potential tool for new applications in biotechnology.

Structural and functional characteristics of xenavidin, the first frog avidin from Xenopus tropicalis.

BACKGROUND: Avidins are proteins with extraordinarily high ligand-binding affinity, a property which is used in a wide array of life science applications. Even though useful for biotechnology and nanotechnology, the biological function of avidins is not fully understood. Here we structurally and functionally characterise a novel avidin named xenavidin, which is to our knowledge the first reported avidin from a frog. RESULTS: Xenavidin was identified from an EST sequence database for Xenopus tropicalis and produced in insect cells using a baculovirus expression system. The recombinant xenavidin was found to be homotetrameric based on gel filtration analysis. Biacore sensor analysis, fluorescently labelled biotin and radioactive biotin were used to evaluate the biotin-binding properties of xenavidin - it binds biotin with high affinity though less tightly than do chicken avidin and bacterial streptavidin. X-ray crystallography revealed structural conservation around the ligand-binding site, while some of the loop regions have a unique design. The location of structural water molecules at the entrance and/or within the ligand-binding site may have a role in determining the characteristic biotin-binding properties of xenavidin. CONCLUSION: The novel data reported here provide information about the biochemically and structurally important determinants of biotin binding. This information may facilitate the discovery of novel tools for biotechnology.

Crystal structure of rhizavidin: insights into the enigmatic high-affinity interaction of an innate biotin-binding protein dimer.

Rhizavidin, from the proteobacterium Rhizobium etli, exhibits high affinity towards biotin but maintains an inherent dimeric quaternary structure and thus, differs from all other known tetrameric avidins. Rhizavidin also differs from the other avidins, since it lacks the characteristic tryptophan residue positioned in the L7,8 loop that plays a crucial role in high-affinity binding and oligomeric stability of the tetrameric avidins. The question is, therefore, how does the dimer exist and how is the high biotin-binding affinity retained? For this purpose, the crystal structures of apo- and biotin-complexed rhizavidin were determined. The structures reveal that the rhizavidin monomer exhibits a topology similar to those of other members of the avidin family, that is, eight antiparallel beta-strands that form the conventional avidin beta-barrel. The quaternary structure comprises the sandwich-like dimer, in which the extensive 1-4 intermonomer interface is intact, but the 1-2 and 1-3 interfaces are nonexistent. Consequently, the biotin-binding site is partially accessible, due to the lack of the tryptophan "lid" that distinguishes the tetrameric structures. In rhizavidin, a disulfide bridge connecting the L3,4 and L5,6 loops restrains the L3,4 loop conformation, leaving the binding-site residues essentially unchanged upon biotin binding. Our study suggests that in addition to the characteristic hydrogen bonding and hydrophobic interactions, the preformed architecture of the binding site and consequent shape complementarity play a decisive role in the high-affinity biotin binding of rhizavidin. The structural description of a novel dimeric avidin-like molecule will greatly contribute to the design of improved and unique avidin derivatives for diversifying the capabilities of avidin-biotin technology.

Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes.

Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys(119)-Cys(299)) and a unique N-linked glycosylation site (Asn(309)) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr(78)) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved.

Bradavidin II from Bradyrhizobium japonicum: a new avidin-like biotin-binding protein.

A gene encoding an avidin-like protein was discovered in the genome of B. japonicum. The gene was cloned to an expression vector and a protein, named bradavidin II, was produced in E. coli. Bradavidin II has an identity of 20-30% and a similarity of 30-40% with previously discovered bradavidin and other avidin-like proteins. It has biochemical characteristics close to those of avidin and streptavidin and binds biotin tightly. In contrast to other tetrameric avidin-like proteins studied to date, bradavidin II has no tryptophan analogous to the W110 in avidin (W120 in streptavidin), thought to be one of the most essential residues for tight biotin-binding. Homology modeling suggests that a proline residue may function analogously to tryptophan in this particular position. Structural elements of bradavidin II such as an interface residue pattern or biotin contact residues could be used as such or transferred to engineered avidin forms to improve or create new tools for biotechnological applications.

Rational modification of ligand-binding preference of avidin by circular permutation and mutagenesis.

Chicken avidin is a key component used in a wide variety of biotechnological applications. Here we present a circularly permuted avidin (cpAvd4-->3) that lacks the loop between beta-strands 3 and 4. Importantly, the deletion of the loop has a positive effect on the binding of 4'-hydroxyazobenzene-2-carboxylic acid (HABA) to avidin. To increase the HABA affinity of cpAvd4-->3 even further, we mutated asparagine 118 on the bottom of the ligand-binding pocket to methionine, which simultaneously caused a significant drop in biotin-binding affinity. The X-ray structure of cpAvd4--> 3(N118M) allows an understanding of the effect of mutation to biotin-binding, whereas isothermal titration calorimetry revealed that the relative binding affinity of biotin and HABA had changed by over one billion-fold between wild-type avidin and cpAvd4-->3(N118M). To demonstrate the versatility of the cpAvd4-->3 construct, we have shown that it is possible to link cpAvd4-->3 and cpAvd5-->4 to form the dual-chain avidin called dcAvd2. These novel avidins might serve as a basis for the further development of self-organising nanoscale avidin building blocks.

Critical importance of loop conformation to avidin-enhanced hydrolysis of an active biotin ester.

The homotetrameric and biotin-binding properties of avidin and streptavidin have been exploited for a myriad of biotechnological applications and theoretical studies. Among the few differences between the two proteins is the capacity of avidin to hydrolyze biotinyl p-nitrophenyl ester (BNP), as opposed to streptavidin, which fully protects the same pseudosubstrate from hydrolysis. Combined mutagenesis and X-ray analysis have been used to attempt to understand this diametric difference in activities. It was found that a charged residue and one of the loops (L3,4) are together responsible for this difference. Recently, the avidin-related analogue AVR4 was found to have an even more pronounced BNP-hydrolysis activity than avidin. Again, the combination of charged residue(s) (Asp39 and/or Arg112) and the rigid conformation of the L3,4 loop was suggested to be responsible for the observed hydrolysis reaction. However, replacement of the latter charged residues in AVR4 resulted in only a modest reduction in hydrolytic activity at most, whereas replacement of the L3,4 loop of avidin with the rigid loop of AVR4 caused a dramatic increase in the activity of avidin. These results clearly demonstrate that the main feature responsible for the observed differences in rates of hydrolysis among the avidins is the conformational status of the L3,4 loop, which imposes conformational constraints on the pseudosubstrate, thereby rendering it susceptible to nucleophilic attack by solvent. In this context, the hydrolytic properties of the avidins reflect enzyme catalysis, in that subtleties in substrate binding are the determining features of catalytic efficiency.

Carbonic anhydrase inhibitors: the very weak inhibitors dithiothreitol, beta-mercaptoethanol, tris(carboxyethyl)phosphine and threitol interfere with the binding of sulfonamides to isozymes II and IX.

The inhibition of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) with dithiothreitol, 2-mercaptoethanol, tris(carboxyethyl)phosphine (reducing agent frequently added to enzyme assay buffers) and threitol has been investigated. The agents were very weak inhibitors of isozymes CA II and CA IX, but unexpectedly, strongly influenced the binding of the low nanomolar sulfonamide inhibitor acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide). Acetazolamide affinity for all investigated CAs diminished orders of magnitude with increasing concentrations of these agents in the assay system. DTT and similar derivatives should not be added to the assay buffers used in monitoring CA activity/inhibition, as they lead to under-estimation of the binding constants, by a mechanism probably involving the formation of ternary complexes.

Brave new (strept)avidins in biotechnology.

Avidin and streptavidin are widely used in (strept)avidin-biotin technology, which is based on their tight biotin-binding capability. These techniques are exceptionally diverse, ranging from simple purification and labeling methods to sophisticated drug pre-targeting and nanostructure-building approaches. Improvements in protein engineering have provided new possibilities to develop tailored protein tools. The (strept)avidin scaffold has been engineered to extend the existing range of applications and to develop new ones. Modifications to (strept)avidins--such as simple amino acid substitutions to reduce biotin binding and alter physico-chemical characters--have recently developed into more sophisticated changes, including chimeric (strept)avidins, topology rearrangements and stitching of non-natural amino acids into the active sites. In this review, we highlight the current status in genetically engineered (strept)avidins and illustrate their versatility as advanced tools in the multiple fields of modern bioscience, medicine and nanotechnology.

Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family.

Rhizobium etli CFN42 is a symbiotic nitrogen-fixing bacterium of the common bean Phaseolus vulgaris. The symbiotic plasmid p42d of R. etli comprises a gene encoding a putative (strept)avidin-like protein, named rhizavidin. The amino acid sequence identity of rhizavidin in relation to other known avidin-like proteins is 20-30%. The amino acid residues involved in the (strept)avidin-biotin interaction are well conserved in rhizavidin. The structural and functional properties of rhizavidin were carefully studied, and we found that rhizavidin shares characteristics with bradavidin, streptavidin and avidin. However, we found that it is the first naturally occurring dimeric protein in the avidin protein family, in contrast with tetrameric (strept)avidin and bradavidin. Moreover, it possesses a proline residue after a flexible loop (GGSG) in a position close to Trp-110 in avidin, which is an important biotin-binding residue. [3H]Biotin dissociation and ITC (isothermal titration calorimetry) experiments showed dimeric rhizavidin to be a high-affinity biotin-binding protein. Its thermal stability was lower than that of avidin; although similar to streptavidin, it was insensitive to proteinase K. The immunological cross-reactivity of rhizavidin was tested with human serum samples obtained from cancer patients exposed to (strept)avidin. No significant cross-reactivity was observed. The biodistribution of the protein was studied by SPECT (single-photon emission computed tomography) imaging in rats. Similarly to avidin, rhizavidin was observed to accumulate rapidly, mainly in the liver. Evidently, rhizavidin could be used as a complement to (strept)avidin in (strept)avidin-biotin technology.

Structure and characterization of a novel chicken biotin-binding protein A (BBP-A).

BACKGROUND: The chicken genome contains a BBP-A gene showing similar characteristics to avidin family genes. In a previous study we reported that the BBP-A gene may encode a biotin-binding protein due to the high sequence similarity with chicken avidin, especially at regions encoding residues known to be located at the ligand-binding site of avidin. RESULTS: Here, we expand the repertoire of known macromolecular biotin binders by reporting a novel biotin-binding protein A (BBP-A) from chicken. The BBP-A recombinant protein was expressed using two different expression systems and purified with affinity chromatography, biochemically characterized and two X-ray structures were solved - in complex with D-biotin (BTN) and in complex with D-biotin D-sulfoxide (BSO). The BBP-A protein binds free biotin with high, "streptavidin-like" affinity (Kd ~ 10-13 M), which is about 50 times lower than that of chicken avidin. Surprisingly, the affinity of BBP-A for BSO is even higher than the affinity for BTN. Furthermore, the solved structures of the BBP-A--BTN and BBP-A--BSO complexes, which share the fold with the members of the avidin and lipocalin protein families, are extremely similar to each other. CONCLUSION: BBP-A is an avidin-like protein having a beta-barrel fold and high affinity towards BTN. However, BBP-A differs from the other known members of the avidin protein family in thermal stability and immunological properties. BBP-A also has a unique ligand-binding property, the ability to bind BTN and BSO at comparable affinities. BBP-A may have use as a novel material in, e.g. modern bio(nano)technological applications.

Controlling quaternary structure assembly: subunit interface engineering and crystal structure of dual chain avidin.

Dual chain avidin (dcAvd) is an engineered avidin form, in which two circularly permuted chicken avidin monomers are fused into one polypeptide chain. DcAvd can theoretically form two different pseudotetrameric quaternary assemblies because of symmetry at the monomer-monomer interfaces. Here, our aim was to control the assembly of the quaternary structure of dcAvd. We introduced the mutation I117C into one of the circularly permuted domains of dcAvd and scanned residues along the 1-3 subunit interface of the other domain. Interestingly, V115H resulted in a single, disulfide locked quaternary assembly of dcAvd, whereas I117H could not guide the oligomerisation process even though it stabilised the protein. The modified dcAvd forms were found to retain their characteristic pseudotetrameric state both at high and low pH, and were shown to bind D-biotin at levels comparable to that of wild-type chicken avidin. The crystal structure of dcAvd-biotin complex at 1.95 Angstroms resolution demonstrates the formation of the functional dcAvd pseudotetramer at the atomic level and reveals the molecular basis for its special properties. Altogether, our data facilitate further engineering of the biotechnologically valuable dcAvd scaffold and gives insights into how to guide the quaternary structure assembly of oligomeric proteins.

Dual-affinity avidin molecules.

A recently reported dual-chain avidin was modified further to contain two distinct, independent types of ligand-binding sites within a single polypeptide chain. Chicken avidin is normally a tetrameric glycoprotein that binds water-soluble d-biotin with extreme affinity (K(d) approximately 10(-15) M). Avidin is utilized in various applications and techniques in the life sciences and in the nanosciences. In a recent study, we described a novel avidin monomer-fusion chimera that joins two circularly permuted monomers into a single polypeptide chain. Two of these dual-chain avidins were observed to associate spontaneously to form a dimer equivalent to the wt tetramer. In the present study, we successfully used this scaffold to generate avidins in which the neighboring biotin-binding sites of dual-chain avidin exhibit two different affinities for biotin. In these novel avidins, one of the two binding sites in each polypeptide chain, the pseudodimer, is genetically modified to have lower binding affinity for biotin, whereas the remaining binding site still exhibits the high-affinity characteristic of the wt protein. The pseudotetramer (i.e., a dimer of dual-chain avidins) has two high and two lower affinity biotin-binding sites. The usefulness of these novel proteins was demonstrated by immobilizing dual-affinity avidin with its high-affinity sites. The sites with lower affinity were then used for affinity purification of a biotinylated enzyme. These "dual-affinity" avidin molecules open up wholly new possibilities in avidin-biotin technology, where they may have uses as novel bioseparation tools, carrier proteins, or nanoscale adapters.

Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells.

The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches.

Tetravalent single-chain avidin: from subunits to protein domains via circularly permuted avidins.

scAvd (single-chain avidin, where two dcAvd are joined in a single polypeptide chain), having four biotin-binding domains, was constructed by fusion of topologically modified avidin units. scAvd showed similar biotin binding and thermal stability properties as chicken avidin. The DNA construct encoding scAvd contains four circularly permuted avidin domains, plus short linkers connecting the four domains into a single polypeptide chain. In contrast with wild-type avidin, which contains four identical avidin monomers, scAvd enables each one of the four avidin domains to be independently modified by protein engineering. Therefore the scAvd scaffold can be used to construct spatially and stoichiometrically defined pseudotetrameric avidin molecules showing different domain characteristics. In addition, unmodified scAvd could be used as a fusion partner, since it provides a unique non-oligomeric structure, which is fully functional with four high-affinity biotin-binding sites. Furthermore, the subunit-to-domain strategy described in the present study could be applied to other proteins and protein complexes, facilitating the development of sophisticated protein tools for applications in nanotechnology and life sciences.

Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family.

BACKGROUND: A chicken egg contains several biotin-binding proteins (BBPs), whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings. RESULTS: Two separate hits showing significant homology for these N-terminal sequences were discovered. For one of these hits, the chromosomal location in the immediate proximity of the avidin gene family was found. Both of these hits encode proteins having high sequence similarity with avidin suggesting that chicken BBPs are paralogous to avidin family. In particular, almost all residues corresponding to biotin binding in avidin are conserved in these putative BBP proteins. One of the found DNA sequences, however, seems to encode a carboxy-terminal extension not present in avidin. CONCLUSION: We describe here the predicted properties of the putative BBP genes and proteins. Our present observations link BBP genes together with avidin gene family and shed more light on the genetic arrangement and variability of this family. In addition, comparative modelling revealed the potential structural elements important for the functional and structural properties of the putative BBP proteins.

Novel avidin-like protein from a root nodule symbiotic bacterium, Bradyrhizobium japonicum.

Bradyrhizobium japonicum is an important nitrogenfixing symbiotic bacterium, which can form root nodules on soybeans. These bacteria have a gene encoding a putative avidin- and streptavidin-like protein, which bears an amino acid sequence identity of only about 30% over the core regions with both of them. We produced this protein in Escherichia coli both as the full-length wild type and as a C-terminally truncated core form and showed that it is indeed a high affinity biotin-binding protein that resembles (strept)avidin structurally and functionally. Because of the considerable dissimilarity in the amino acid sequence, however, it is immunologically very different, and polyclonal rabbit and human antibodies toward (strept)avidin did not show significant cross-reactivity with it. Therefore this new avidin, named bradavidin, facilitates medical treatments such as targeted drug delivery, gene therapy, and imaging by offering an alternative tool for use if (strept)avidin cannot be used, because of a deleterious patient immune response for example. In addition to its medical value, bradavidin can be used both in other applications of avidin-biotin technology and as a source of new ideas when creating engineered (strept)avidin forms by changing or combining the desired parts, interface patterns, or specific residues within the avidin protein family. Moreover, the unexpected discovery of bradavidin indicates that the group of new and undiscovered bacterial avidin-like proteins may be both more diverse and more common than hitherto thought.

A multipurpose vector system for the screening of libraries in bacteria, insect and mammalian cells and expression in vivo.

We have constructed a novel tetra-promoter vector (pBVboostFG) system that enables screening of gene/cDNA libraries for functional genomic studies. The vector enables an all-in-one strategy for gene expression in mammalian, bacterial and insect cells and is also suitable for direct use in vivo. Virus preparation is based on an improved mini Tn7 transpositional system allowing easy and fast production of recombinant baculoviruses with high diversity and negligible background. Cloning of the desired DNA fragments or libraries is based on the recombination system of bacteriophage lambda. As an example of the utility of the vector, genes or cDNAs of 18 different proteins were cloned into pBVboostFG and expressed in different hosts. As a proof-of-principle of using the vector for library screening, a chromophoric Thr65-Tyr-Gly67-stretch of enhanced green fluorescent protein was destroyed and subsequently restored by novel PCR strategy and library screening. The pBVboostFG enables screening of genome-wide libraries, thus making it an efficient new platform technology for functional genomics.

Design and construction of highly stable, protease-resistant chimeric avidins.

The chicken avidin gene family consists of avidin and seven separate avidin-related genes (AVRs) 1-7. Avidin protein is a widely used biochemical tool, whereas the other family members have only recently been produced as recombinant proteins and characterized. In our previous study, AVR4 was found to be the most stable biotin binding protein thus far characterized (T(m) = 106.4 degrees C). In this study, we studied further the biotin-binding properties of AVR4. A decrease in the energy barrier between the biotin-bound and unbound state of AVR4 was observed when compared with that of avidin. The high resolution structure of AVR4 facilitated comparison of the structural details of avidin and AVR4. In the present study, we used the information obtained from these comparative studies to transfer the stability and functional properties of AVR4 to avidin. A chimeric avidin protein, ChiAVD, containing a 21-amino acid segment of AVR4 was found to be significantly more stable (T(m) = 96.5 degrees C) than native avidin (T(m) = 83.5 degrees C), and its biotin-binding properties resembled those of AVR4. Optimization of a crucial subunit interface of avidin by an AVR4-inspired point mutation, I117Y, significantly increased the thermostability of the avidin mutant (T(m) = 97.5 degrees C) without compromising its high biotin-binding properties. By combining these two modifications, a hyperthermostable ChiAVD(I117Y) was constructed (T(m) = 111.1 degrees C). This study provides an example of rational protein engineering in which another member of the protein family has been utilized as a source in the optimization of selected properties.