Interdomain Linker Region Research Articles

Mapping of the Vitronectin-binding Site on the Urokinase Receptor: INVOLVEMENT OF A COHERENT RECEPTOR INTERFACE CONSISTING OF RESIDUES FROM BOTH DOMAIN I AND THE FLANKING INTERDOMAIN LINKER REGION

The urokinase-type plasminogen activator receptor (uPAR) has been implicated as a modulator of several biochemical processes that are active during tumor invasion and metastasis, e.g. extracellular proteolysis, cell adhesion, and cell motility. The structural basis for the high affinity interaction between the urokinase-type plasminogen activator (uPA) and uPAR, which focuses cell surface-associated plasminogen activation in vivo, is now thoroughly characterized by site-directed mutagenesis studies and x-ray crystallography. In contrast, the structural basis for the interaction between uPAR and the extracellular matrix protein vitronectin, which is involved in the regulation of cell adhesion and motility, remains to be clarified. In this study, we have identified the functional epitope on uPAR that is responsible for its interaction with the full-length, extended form of vitronectin by using a comprehensive alanine-scanning library of purified single-site uPAR mutants (244 positions tested). Interestingly, the five residues identified as "hot spots" for vitronectin binding form a contiguous epitope consisting of two exposed loops connecting the central fourstranded beta-sheet in uPAR domain I (Trp(32), Arg(58), and Ile(63)) as well as a proximal region of the flexible linker peptide connecting uPAR domains I and II (Arg(91) and Tyr(92)). This binding topology provides the molecular basis for the observation that uPAR can form a ternary complex with uPA and vitronectin. Furthermore, it raises the intriguing possibility that the canonical receptor and inhibitor for uPA (uPAR and PAI-1) may have reached a convergent solution for binding to the somatomedin B domain of vitronectin.

Structure of the Subunit Binding Domain and Dynamics of the Di-domain Region from the Core of Human Branched Chain α-Ketoacid Dehydrogenase Complex

The homo-24-meric dihydrolipoyl transacylase (E2) scaffold of the human branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) contains the lipoyl-bearing domain (hbLBD), the subunit-binding domain (hbSBD) and the inner core domain that are linked to carry out E2 functions in substrate channeling and recognition. In this study, we employed NMR techniques to determine the structure of hbSBD and dynamics of several truncated constructs from the E2 component of the human BCKDC, including hbLBD (residues 1-84), hbSBD (residues 111-149), and a di-domain (hbDD) (residues 1-166) comprising hbLBD, hbSBD and the interdomain linker. The solution structure of hbSBD consists of two nearly parallel helices separated by a long loop, similar to the structures of the SBD isolated from other species, but it lacks the short 3(10) helix. The NMR results show that the structures of hbLBD and hbSBD in isolated forms are not altered by the presence of the interdomain linker in hbDD. The linker region is not entirely exposed to solvent, where amide resonances associated with approximately 50% of the residues are observable. However, the tethering of these two domains in hbDD significantly retards the overall rotational correlation times of hbLBD and hbSBD, changing from 5.54 ns and 5.73 ns in isolated forms to 8.37 ns and 8.85 ns in the linked hbDD, respectively. We conclude that the presence of the interdomain linker restricts the motional freedom of the hbSBD more significantly than hbLBD, and that the linker region likely exists as a soft rod rather than a flexible string in solution.

Crystal Structure of Penicillin-binding Protein 1a (PBP1a) Reveals a Mutational Hotspot Implicated in β-Lactam Resistance in Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen whose infections have been treated with beta-lactam antibiotics for over 60 years, but the proliferation of strains that are highly resistant to such drugs is a problem of worldwide concern. Beta-lactams target penicillin-binding proteins (PBPs), membrane-associated enzymes that play essential roles in the peptidoglycan biosynthetic process. Bifunctional PBPs catalyze both the polymerization of glycan chains (glycosyltransfer) and the cross-linking of adjacent pentapeptides (transpeptidation), while monofunctional enzymes catalyze only the latter reaction. Although S. pneumoniae has six PBPs, only three (PBP1a, PBP2x, PBP2b) are major resistance determinants, with PBP1a being the only bifunctional enzyme. PBP1a plays a key role in septum formation during the cell division cycle and its modification is essential for the development of high-level resistance to penicillins and cephalosporins. The crystal structure of a soluble form of pneumococcal PBP1a (PBP1a*) has been solved to 2.6A and reveals that it folds into three domains. The N terminus contains a peptide from the glycosyltransfer domain bound to an interdomain linker region, followed by a central, transpeptidase domain, and a small C-terminal unit. An analysis of PBP1a sequences from drug-resistant clinical strains in light of the structure reveals the existence of a mutational hotspot at the entrance of the catalytic cleft that leads to the modification of the polarity and accessibility of the mutated PBP1a active site. The presence of this hotspot in all variants sequenced to date is of key relevance for the development of novel antibiotherapies for the treatment of beta-lactam-resistant pneumococcal strains.

191. Identification of a Novel Functional Domain in the Sleeping Beauty Transposase: Towards Alleviating the Restriction of SB Overproduction Inhibition

DNA transposons such as Sleeping Beauty (SB) have been shown to be effective vectors for gene therapy. However, one limitation of the SB system is its regulation by a phenomenon known as overproduction inhibition (OPI), in which elevated concentrations of transposase enzyme inhibit the transposition reaction. In an attempt to characterize the mechanism behind this process, we studied the structure/function relationship of the wild-type SB10 protein|[mdash]|focusing on the region linking the N-terminal DNA binding domain and the C-terminal catalytic domain. This area of SB10 is not only highly conserved (55-60%) among the Tc1/Mariner family of transposases, but has also been previously implicated in adversely affecting SB's DNA binding ability. We first tested whether regions downstream of the transposase N-terminus contribute to SB10 DNA binding activity by analyzing various SB deletion mutants in a yeast one-hybrid assay for transposase-transposon interactions. Results show that the ability of the protein to bind transposon DNA increases exponentially in a manner that is proportional with the size of the C-terminal deletion. Mutational analyses of this inter-domain linker region indicate that it is important for transposase function since nearly all mutants analyzed were either completely inactive or 2-3 fold hyperactive in a quantitative transposition assay. To ascertain whether this newly-identified functional domain contributes to OPI, SB10 and five hyperactive mutants were assayed for transposition activity at increasing transposase:transposon ratios. All mutants demonstrated altered OPI titration curves as compared to SB10, with two distinct mutant classes emerging. Class 1 mutants yielded a curve which was shifted towards higher transposase:transposon ratios, but had the same maximum activity as the SB10 curve. In contrast, Class 2 mutants had transposition levels only reaching |[sim]|50% that of SB10. However, these curves were also shifted toward higher amounts of transposase. Based on the similar shift in curves for both types of mutants, we concluded that mutations in the linker domain resulted in an increased OPI threshold. To elucidate the molecular mechanism behind this change in SB regulation, we tested the binding abilities of these linker domain mutants using our yeast one-hybrid assay. We found that all the inactive transposase mutants were able to bind transposon DNA at least as well as SB10 and some of hyperactive mutants exhibited up to a 4-fold change in DNA binding ability. Though some of the mutants did show a dramatic change in transposon binding, we observed no obvious overall correlation between binding and transpositional regulation. However, based on an altered OPI curve of an N-terminal transposase mutant with known increased affinity for the transposon, we suspect that some relationship between binding and regulation exists. Although we do not as yet completely understand the mechanism behind OPI, our results indicate that it is possible to remove some of the stringency of SB self-inhibition. Through continued study of this regulatory process, we hope to eventually overcome this obstacle and improve the efficiency and utility of SB as a vehicle for gene transfer.

Active site restructuring regulates ligand recognition in class A penicillin-binding proteins

Bacterial cell division is a complex, multimolecular process that requires biosynthesis of new peptidoglycan by penicillin-binding proteins (PBPs) during cell wall elongation and septum formation steps. Streptococcus pneumoniae has three bifunctional (class A) PBPs that catalyze both polymerization of glycan chains (glycosyltransfer) and cross-linking of pentapeptidic bridges (transpeptidation) during the peptidoglycan biosynthetic process. In addition to playing important roles in cell division, PBPs are also the targets for beta-lactam antibiotics and thus play key roles in drug-resistance mechanisms. The crystal structure of a soluble form of pneumococcal PBP1b (PBP1b*) has been solved to 1.9 A, thus providing previously undescribed structural information regarding a class A PBP from any organism. PBP1b* is a three-domain molecule harboring a short peptide from the glycosyltransferase domain bound to an interdomain linker region, the transpeptidase domain, and a C-terminal region. The structure of PBP1b* complexed with beta-lactam antibiotics reveals that ligand recognition requires a conformational modification involving conserved elements within the cleft. The open and closed structures of PBP1b* suggest how class A PBPs may become activated as novel peptidoglycan synthesis becomes necessary during the cell division process. In addition, this structure provides an initial framework for the understanding of the role of class A PBPs in the development of antibiotic resistance.

A role for the interdomain linker region of the Escherichia coli CytR regulator in repression complex formation.

Regulatory complexes formed by the CytR repressor protein and the cAMP receptor protein (CRP) prevent transcription initiation from several promoters in Escherichia coli. The formation of the complexes is mediated by protein-DNA interactions and protein-protein interactions between the two regulators. Interestingly, co-binding with CRP has a profound effect on the configuration of the DNA-binding targets preferred by CytR. When binding to DNA by itself, CytR binds preferentially to two octamer repeats in direct or inverted orientation, and separated by 2 bp. However, in the presence of CRP, CytR recognizes inverted repeats separated by 10-13 bp, or direct repeats separated by 1 bp. A fixed orientation of at least one CytR octamer repeat in close proximity to a CRP-binding target is a common architectural feature at promoters optimised for repression complex formation. These observations suggest that CRP alters the DNA-binding mode of CytR. Here, we have investigated the CRP-induced changes in CytR by protein footprinting and alanine-scanning mutagenesis. Our data suggest that a flexible interdomain linker region in CytR, connecting the DNA-binding domain to the dimerization domain allows the repressor protein to interact with DNA-binding sites in a highly relaxed manner, as shown previously, and plays an active role in transcription regulation. Thus, the interactions between CRP, CytR and DNA within the repression complex appear to be more extensive than anticipated. The results support and extend the view that the high degree of adaptability observed in the CytR/CRP regulatory system is obtained though multiple adjustable interactions between the implicated factors.

Analysis of the retrovirus capsid interdomain linker region

In structural studies, the retrovirus capsid interdomain linker region has been shown as a flexible connector between the CA N-terminal domain and its C-terminal domain. To analyze the function of the linker region, we have examined the effects of three Moloney murine leukemia virus (M-MuLV) capsid linker mutations/variations in vivo, in the context of the full-length M-MuLV structural precursor protein (PrGag). Two mutations, A1SP and A5SP, respectively, inserted three and seven additional codons within the linker region to test the effects of increased linker lengths. The third variant, HIV/Mo, represented a chimeric HIV-1/M-MuLV PrGag protein, fused at the linker region. When expressed in cells, the three variants reduced the efficiency of virus particle assembly, with PrGag proteins and particles accumulating at the cellular plasma membranes. Although PrGag recognition of viral RNA was not impaired, the capsid linker variant particles were abnormal, with decreased stabilities, anomalous densities, and aberrant multiple lobed and tubular morphologies. Additionally, rather than crosslinking as PrGag dimers, particle-associated A1SP, A5SP, and HIV/Mo proteins showed an increased propensity to crosslink as trimers. Our results suggest that a wild-type retrovirus capsid linker region is required for the proper alignment of capsid protein domains.

Zinc- and pH-dependent conformational transition in a putative interdomain linker region of the influenza virus matrix protein M1.

The matrix protein M1 of influenza A virus forms a shell beneath the viral envelope and sustains the virion architecture by interacting with other viral components. A structural change of M1 upon acidification of the virion interior in an early stage of virus infection is considered to be a key step to virus uncoating. We examined the structure of a 28-mer peptide (M1Lnk) representing a putative linker region between the N- and C-terminal domains of M1 by using circular dichroism, Raman, and absorption spectroscopy. M1Lnk assumes an alpha-helical structure in a mildly hydrophobic environment irrespective of pH, being consistent with the X-ray crystal structures of an N-terminal fragment of M1 at pH 7 and 4. In the presence of Zn(2+), on the other hand, M1Lnk takes a partially unfolded conformation at neutral pH with a tetrahedral coordination of two Cys residues and two His residues to a Zn(2+) ion in the central part of the peptide. Upon acidification, the peptide releases the Zn(2+) ion and refolds into the alpha-helix-rich structure with a midpoint of transition at pH 5.9. The pH-dependent conformational transition of M1Lnk strongly suggests that the interdomain linker region of M1 also undergoes a pH-dependent unfolding-refolding transition in the presence of Zn(2+). A small but significant portion of the M1 protein is bound to Zn(2+) in the virion, and the Zn(2+)-bound M1 molecule may play a special role in virus uncoating by changing the disposition of the N- and C-terminal domains upon acidification of the virion interior.

Differential Affinity and Cooperativity Functions of the Amino-terminal 70 Residues of λ Integrase

The site-specific recombinase (Int) of bacteriophage λ is a heterobivalent DNA-binding protein that binds two different classes of DNA-binding sites within its recombination target sites. The several functions of Int are apportioned between a large carboxy-terminal domain that cleaves and ligates DNA at each of its four “core-type” DNA-binding sites and a small amino-terminal domain, whose primary function is binding to each of its five “arm-type” DNA sites, which are distant from the core region. Int bridges between the two classes of binding sites are facilitated by accessory DNA-bending proteins that along with Int comprise higher-order recombinogenic complexes. We show here that although the 64 amino-terminal residues of Int bind efficiently to a single arm site, this protein cannot form doubly bound complexes on adjacent arm sites. However, 1-70 Int does show the same cooperative binding to adjacent arm sites as the full length protein. We also found that 1-70 Int specifies cooperative interactions with the accessory protein Xis when the two are bound to their adjacent cognate sites P2 and X1, respectively. To complement the finding that these two amino-terminal domain functions (along with arm DNA binding) are all specified by residues 1–70, we determined that Thr75 is the first residue of the minimal carboxy-terminal domain, thereby identifying a specific interdomain linker region. We have measured the affinity constants for Int binding to each of the five arm sites and the cooperativity factors for Int binding to the two pairs of adjacent arm sites, and we have identified several DNA structural features that contribute to the observed patterns of Int binding to arm sites. Taken together, the results highlight several interesting features of arm DNA binding that invite speculation about additional levels of complexity in the regulation of λ site-specific recombination.

The Interdomain Region of Dengue NS5 Protein That Binds to the Viral Helicase NS3 Contains Independently Functional Importin β1 and Importin α/β-Recognized Nuclear Localization Signals

Dengue virus NS5 protein is a multifunctional RNA-dependent RNA polymerase that is essential for virus replication. We have shown previously that the 37- amino acid interdomain spacer sequence (residues (369)X(2)KKX(14)KKKX(11)RKX(3)405) of Dengue2 NS5 contains a functional nuclear localization signal (NLS). In this study, beta-galactosidase fusion proteins carrying point mutations of the positively charged residues or truncations of the interdomain linker region (residues 369-389 or residues 386-405) were analyzed for nuclear import and importin binding activities to show that the N-terminal part of the linker region (residues 369-389, a/bNLS) is critical for nuclear localization and is recognized with high affinity by the conventional NLS-binding importin alpha/beta heterodimeric nuclear import receptor. We also show that the importin beta-binding site (residues 320-368, bNLS) adjacent to the a/bNLS, previously identified by yeast two-hybrid analysis, is functional as an NLS, recognized with high affinity by importin beta, and able to target beta-galactosidase to the nucleus. Intriguingly, the bNLS is highly conserved among Dengue and related flaviviruses, implying a general role for the region and importin beta in the infectious cycle.

Solution structure of the human Hck SH3 domain and identification of its ligand binding site

SH3 domains are protein binding domains that occur widely among signal transduction proteins. Here, we present the NMR-determined solution structure of the SH3 domain from the cytoplasmic protein tyrosine kinase, Hck. Hck is involved in a number of cell signal transduction pathways, frequently in pathways associated with immune response. SH3 domains bind proteins via a left-handed polyproline type II helix on the target protein. We have assessed the structural impact of binding to a ligand through addition of a peptide corresponding to a proline-rich region of a Hck target, the GTPase activating protein of the Ras pathway. Ligand binding effects small structural changes and stabilizes the SH3 domain structure. Also, we have compared the solution structure of the Hck SH3 domain to the crystal structure of Hck, in which the SH3 domain exhibits an intramolecular binding to an interdomain linker region. These structures are interpreted as the apo- and holo- forms of the Hck SH3 domain.

Properties of a single-chain antibody containing different linker peptides.

Single-chain antibodies were constructed using six different linker peptides to join the VH and VL domains of an anti-2-phenyloxazolone (Ox) antibody. Four of the linker peptides originated from the interdomain linker region of the fungal cellulase CBHI and consisted of 28, 11, six and two amino acid residues. The two other linker peptides used were the (GGGGS)3 linker with 15 amino acid residues and a modified IgG2b hinge peptide with 22 residues. Proteolytic stability and Ox binding properties of the six different scFv derivatives produced in Escherichia coli were investigated and compared with those of the corresponding Fv fragment containing no joining peptide between the V domains. The hapten binding properties of different antibody fragments were studied by ELISA and BIAcoreTM. The interdomain linker peptide improved the hapten binding properties of the antibody fragment when compared with Fv fragment, but slightly increased its susceptibility to proteases. Single-chain antibodies with short CBHI linkers of 11, six and two residues had a tendency to form multimers which led to a higher apparent affinity. The fragments with linkers longer than 11 residues remained monomeric.

Reaching Out Locating and Lengthening the Interdomain Linker in AraC Protein

A genetic method was developed to determine, in proteins, areas which are tolerant of insertions and deletions. Attractive candidates for these areas are linker regions. Such a region was found to include positions 171 to 178 in the Escherichia coli regulatory protein AraC. Independent biochemical methods identified amino acid residues 11 to 170 as the minimal dimerization domain of AraC, and amino acid residues 178 to 286 out of the 291 residue protein as the minimal DNA-binding domain. Hence, by both the genetic and biochemical approaches, the interdomain linking region was determined to include amino acid residues 171 to 177. The properties of altered proteins were examined using templates with AraC half-sites more widely separated than in the wild-type case. Both AraC protein containing an insertion in the interdomain linker region and a protein consisting of the minimal functional dimerization and DNA-binding domains separated by a 39 amino acid residue linker were able to bind to and function on such a DNA site. In vitro, the proteins with longer linkers bound substantially more stably than wild-type AraC to the DNA containing half-sites for AraC separated by an extra two helical turns of DNA. In vivo on an ara promoter with the more widely separated AraC half-sites, the proteins could activate transcription much better than wild-type AraC.

Reaching Out: Locating and Lengthening the Interdomain Linker in AraC Protein

A genetic method was developed to determine, in proteins, areas which are tolerant of insertions and deletions. Attractive candidates for these areas are linker regions. Such a region was found to include positions 171 to 178 in the Escherichia coli regulatory protein AraC. Independent biochemical methods identified amino acid residues 11 to 170 as the minimal dimerization domain of AraC, and amino acid residues 178 to 286 out of the 291 residue protein as the minimal DNA-binding domain. Hence, by both the genetic and biochemical approaches, the interdomain linking region was determined to include amino acid residues 171 to 177. The properties of altered proteins were examined using templates with AraC half-sites more widely separated than in the wild-type case. Both AraC protein containing an insertion in the interdomain linker region and a protein consisting of the minimal functional dimerization and DNA-binding domains separated by a 39 amino acid residue linker were able to bind to and function on such a DNA site. In vitro, the proteins with longer linkers bound substantially more stably than wild-type AraC to the DNA containing half-sites for AraC separated by an extra two helical turns of DNA. In vivo on an ara promoter with the more widely separated AraC half-sites, the proteins could activate transcription much better than wild-type AraC.

An active single-chain antibody containing a cellulase linker domain is secreted by Escherichia coli

Single-chain antibodies consist of the variable, antigen-binding domains of antibodies joined to a continuous polypeptide by genetically engineered peptide linkers. We have used the flexible interdomain linker region of a fungal cellulase to link together the variable domains of an anti-2-phenyloxazolone IgG1 and show here that the resulting single-chain antibody is efficiently secreted and released to the culture medium of Escherichia coli. The yield of affinity-purified single-chain antibody is 1-2 mg/l of culture medium and its affinity and stability are comparable to those of the corresponding native IgG.

Essential and non-essential domains in the Bradyrhizobium japonicum NifA protein: identification of indispensable cysteine residues potentially involved in redox reactivity and/or metal binding.

The amino acid sequence of the Bradyrhizobium japonicum nitrogen fixation regulatory protein NifA, as derived from the nucleotide sequence of the nifA gene, was aligned to the corresponding protein sequences from Klebsiella pneumoniae, Rhizobium meliloti and Rhizobium leguminosarum biovar viciae. High conservation was found in the central domain and in the COOH-terminal, putative DNA binding domain, whereas very little homology was present within the first 250 amino acids from the NH2-terminus. Upon deletion of the first 218 amino acids (37% of the protein) and expression of the remainder as a Cat'-'NifA hybrid protein, a fully active, nif-specific transcriptional activator protein was obtained which also retained oxygen sensitivity, a characteristic property of the wild-type B. japonicum NifA protein. In contrast, an unaltered COOH-terminal domain was required for an active NifA protein. Between the central and the DNA binding domains, a so-called interdomain linker region was identified which was conserved in all rhizobial species but missing in the K.pneumoniae NifA protein. Two conserved cysteine residues in this region were changed to serine residues, by oligonucleotide-directed mutagenesis. This resulted in absolutely inactive NifA mutant proteins. Similar null phenotypes were obtained by altering two closely adjacent cysteine residues in the central domain to serine residues. Nif gene activation in vivo by the B.japonicum NifA protein, but not by the K.pneumoniae NifA protein, was sensitive to treatment with chelating agents, and this inhibition could be overcome by the addition of divalent metal ions. On the basis of these observations and previous data on oxygen sensitivity we raise the hypothesis that at least some, if not all, of the four essential cysteine residues may be involved in oxygen reactivity or metal binding or both.