Consequences of inducing intrinsic disorder in a high-affinity protein-protein interaction.

The kinetic and thermodynamic consequences of intrinsic disorder in protein-protein recognition are controversial. We address this by inducing one partner of the high-affinity colicin E3 rRNase domain-Im3 complex (K(d) ≈ 10(-12) M) to become an intrinsically disordered protein (IDP). Through a variety of biophysical measurements, we show that a single alanine mutation at Tyr507 within the hydrophobic core of the isolated colicin E3 rRNase domain causes the enzyme to become an IDP (E3 rRNase(IDP)). E3 rRNase(IDP) binds stoichiometrically to Im3 and forms a structure that is essentially identical to the wild-type complex. However, binding of E3 rRNase(IDP) to Im3 is 4 orders of magnitude weaker than that of the folded rRNase, with thermodynamic parameters reflecting the disorder-to-order transition on forming the complex. Critically, pre-steady-state kinetic analysis of the E3 rRNase(IDP)-Im3 complex demonstrates that the decrease in affinity is mostly accounted for by a drop in the electrostatically steered association rate. Our study shows that, notwithstanding the advantages intrinsic disorder brings to biological systems, this can come at severe kinetic and thermodynamic cost.

Structure of the virulence-associated protein VapD from the intracellular pathogen Rhodococcus equi.

Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded ß-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-ß-D-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the ß-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other ß-barrel proteins.

Evidence for steric regulation of fibrinogen binding to Staphylococcus aureus fibronectin-binding protein A (FnBPA).

The adjacent fibrinogen (Fg)- and fibronectin (Fn)-binding sites on Fn-binding protein A (FnBPA), a cell surface protein from Staphylococcus aureus, are implicated in the initiation and persistence of infection. FnBPA contains a single Fg-binding site (that also binds elastin) and multiple Fn-binding sites. Here, we solved the structure of the N2N3 domains containing the Fg-binding site of FnBPA in the apo form and in complex with a Fg peptide. The Fg binding mechanism is similar to that of homologous bacterial proteins but without the requirement for "latch" strand residues. We show that the Fg-binding sites and the most N-terminal Fn-binding sites are nonoverlapping but in close proximity. Although Fg and a subdomain of Fn can form a ternary complex on an FnBPA protein construct containing a Fg-binding site and single Fn-binding site, binding of intact Fn appears to inhibit Fg binding, suggesting steric regulation. Given the concentrations of Fn and Fg in the plasma, this mechanism might result in targeting of S. aureus to fibrin-rich thrombi or elastin-rich tissues.

Staphylococcal biofilm-forming protein has a contiguous rod-like structure.

Staphylococcus aureus and Staphylococcus epidermidis form communities (called biofilms) on inserted medical devices, leading to infections that affect many millions of patients worldwide and cause substantial morbidity and mortality. As biofilms are resistant to antibiotics, device removal is often required to resolve the infection. Thus, there is a need for new therapeutic strategies and molecular data that might assist their development. Surface proteins S. aureus surface protein G (SasG) and accumulation-associated protein (S. epidermidis) promote biofilm formation through their "B" regions. B regions contain tandemly arrayed G5 domains interspersed with approximately 50 residue sequences (herein called E) and have been proposed to mediate intercellular accumulation through Zn(2+)-mediated homodimerization. Although E regions are predicted to be unstructured, SasG and accumulation-associated protein form extended fibrils on the bacterial surface. Here we report structures of E-G5 and G5-E-G5 from SasG and biophysical characteristics of single and multidomain fragments. E sequences fold cooperatively and form interlocking interfaces with G5 domains in a head-to-tail fashion, resulting in a contiguous, elongated, monomeric structure. E and G5 domains lack a compact hydrophobic core, and yet G5 domain and multidomain constructs have thermodynamic stabilities only slightly lower than globular proteins of similar size. Zn(2+) does not cause SasG domains to form dimers. The work reveals a paradigm for formation of fibrils on the 100-nm scale and suggests that biofilm accumulation occurs through a mechanism distinct from the "zinc zipper." Finally, formation of two domains by each repeat (as in SasG) might reduce misfolding in proteins when the tandem arrangement of highly similar sequences is advantageous.

The membrane proteins SiaQ and SiaM form an essential stoichiometric complex in the sialic acid tripartite ATP-independent periplasmic (TRAP) transporter SiaPQM (VC1777-1779) from Vibrio cholerae.

Tripartite ATP-independent periplasmic (TRAP) transporters are widespread in bacteria but poorly characterized. They contain three subunits, a small membrane protein, a large membrane protein, and a substrate-binding protein (SBP). Although the function of the SBP is well established, the membrane components have only been studied in detail for the sialic acid TRAP transporter SiaPQM from Haemophilus influenzae, where the membrane proteins are genetically fused. Herein, we report the first in vitro characterization of a truly tripartite TRAP transporter, the SiaPQM system (VC1777-1779) from the human pathogen Vibrio cholerae. The active reconstituted transporter catalyzes unidirectional Na(+)-dependent sialic acid uptake having similar biochemical features to the orthologous system in H. influenzae. However, using this tripartite transporter, we demonstrate the tight association of the small, SiaQ, and large, SiaM, membrane proteins that form a 1:1 complex. Using reconstituted proteoliposomes containing particular combinations of the three subunits, we demonstrate biochemically that all three subunits are likely to be essential to form a functional TRAP transporter.

Comparative assessment of different histidine-tags for immobilization of protein onto surface plasmon resonance sensorchips.

Surface plasmon resonance (SPR) is widely used to assess the kinetics and thermodynamics of binding of two molecules. The major challenge is immobilization of one molecule onto the sensorchip for robust detection of binding of the other molecule. We have compared a number of immobilization strategies for noncovalent attachment of an example protein (the substrate binding protein SiaP) by hexa-histidine (His), deca-His, and double-His tags to a nickel-nitrilotriacetic acid (NTA) surface. The stability of immobilization was assessed, and the binding of two low molecular weight ligands, Neu5Ac and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), at different temperatures studied. The hexa-His tagged SiaP washed off from the surface too rapidly for ligand binding to be measured reliably. Systematic variation of chip loading identified conditions under which the deca-His tagged SiaP could generate reliable results. The double-His tagged protein performed as well as covalently attached deca-His tagged protein at 15, 25, and 35 °C. The observed ligand binding kinetics were comparable for all immobilization strategies, and thermodynamic values calculated from SPR are in agreement with solution-based isothermal titration calorimetry measurements. Extended trials suggest that covalent attachment is preferable for screening campaigns, whereas the double-His-tag strategy allows rapid regeneration of the chip, for example, when tight binding compounds are assessed.

Mutations in subdomain B of the minichromosome maintenance (MCM) helicase affect DNA binding and modulate conformational transitions.

Minichromosome maintenance (MCM) proteins are believed to provide the replicative helicase activity in eukaryotes and archaea. The single MCM orthologue from Methanothermobacter thermautotrophicus (MthMCM) has been extensively characterized as a model of the eukaryotic heterohexameric MCM complex. MthMCM forms high molecular weight complexes in solution consistent with a dodecamer. Visualization of this complex by electron microscopy suggests that single and double heptameric or hexameric rings can form. We have mutated two arginine residues (Arg-137, Arg-160) in the N-terminal subdomain B of MthMCM based on their apparent potential to form inter-ring hydrogen bonds. Both the single R137A and the double R137A,R160A mutants were characterized by a combination of biophysical, biochemical, and electron microscopy techniques. Biophysical analysis coupled with electron microscopy studies shows that the R137A mutant forms a double heptameric ring, whereas the R137A,R160A protein assembles as a single heptamer. They both show a defect in DNA binding and a concomitant conformational change in subdomain A, with the double mutant displaying significant defects in helicase activity as well. We propose a model in which MCM loading and the subsequent activation of the helicase activity involve a conformational transition that is connected to a DNA binding event.

Two HlyIIR dimers bind to a long perfect inverted repeat in the operator of the hemolysin II gene from Bacillus cereus.

HlyIIR is a negative transcriptional regulator of hemolysin II gene from B. cereus. It binds to a long DNA perfect inverted repeat (44bp) located upstream the hlyII gene. Here we show that HlyIIR is dimeric in solution and in bacterial cells. No protein-protein interactions between dimers and no significant modification of target DNA conformation upon complex formation were observed. Two HlyIIR dimers were found to bind to native operator independently with Kd level in the nanomolar range. The minimal HlyIIR binding site was identified as a half of the long DNA perfect inverted repeat.

Biochemical characterization of RAR1 cysteine- and histidine-rich domains (CHORDs): a novel class of zinc-dependent protein-protein interaction modules.

Disease resistance in plants requires the activation of defense signaling pathways to prevent the spread of infection. The protein Required for Mla12 Resistance (RAR1) is a component of such pathways, which contains cysteine- and histidine-rich domains (CHORDs) that bind zinc. CHORDs are 60 amino acid domains, usually arranged in tandem, found in almost all eukaryotes, where they are involved in processes ranging from pressure sensing in the heart to maintenance of diploidy in fungi, and exhibit distinct protein-protein interaction specificity. In the case of RAR1, CHORD-I is known to interact with heat-shock protein 90 (HSP90) and CHORD-II is known to interact with the Suppressor of the G2 allele of Skp1 (SGT1). The focus of this work on RAR1 from barley and Arabidopsis was to address the paucity of biochemical information on RAR1 and its constituent CHORDs, particularly the role of the metal ion. Sedimentation experiments indicated RAR1 to be an extended monomer in solution with few intramolecular interactions. This was reinforced by denaturation experiments, where little difference between the stability of the individual domains and intact RAR1 could be detected by intrinsic tryptophan fluorescence. Electrospray ionization-mass spectrometry and atomic absorption showed that, contrary to previous reports, RAR1 binds five zinc ions; each CHORD binds two, and the plant-specific, 20 amino acid cysteine- and histidine-containing motif (CCCH motif) located between the two CHORDs binds the fifth. Fluorescence, ultraviolet circular dichroism (UV CD), and nuclear magnetic resonance (NMR) spectroscopy further demonstrated that zinc ions are essential for maintaining CHORD structure. Finally, we used isothermal titratrion colarimetry to show that zinc is essential for the specific binding interactions of CHORD-II with SGT1. Our study provides the first biochemical and biophysical data on the zinc metalloprotein RAR1, defines its metal stoichiometry and that of its constituent CHORDs, and reveals that the metal ions are essential for structural integrity and specific protein-protein associations.

Expression and characterisation of a major c-type cytochrome encoded by gene kustc0563 from Kuenenia stuttgartiensis as a recombinant protein in Escherichia coli.

The purification of small quantities of a major small c-type cytochrome from the anammox bacterium Kuenenia stuttgartiensis has recently been reported. In order to characterise this protein further we have expressed the gene encoding this cytochrome in Escherichia coli and have purified the protein to homogeneity. The protein is directed to the E. coli periplasm using its native signal sequence suggesting that it may be translocated via a Sec-type system in K. stuttgartiensis. The cytochrome has the visible spectroscopic properties typical of a low-spin c-type cytochrome, but these spectroscopic features broaden in high salt solutions. The oxidised cytochrome was able to bind the ligands NO and cyanide. A redox potential of +230 mV suggests that the protein is suitable to act as an electron carrier protein that may be involved in the respiratory chain between hydrazine oxidation and the reduction of nitrite. The predicted protein sequence for the cytochrome suggests it to be a predominantly alpha-helical protein, and this is supported by circular dichroism.

Heterologous overexpression and purification of cytochrome c' from Rhodobacter capsulatus and a mutant (K42E) in the dimerization region. Mutation does not alter oligomerization but impacts the heme iron spin state and nitric oxide binding properties.

Rhodobacter capsulatus cytochrome c' (RCCP) has been overexpressed in Escherichia coli, and its spectroscopic and ligand-binding properties have been investigated. It is concluded that the heterologously expressed protein is assembled correctly, as judged by UV-vis absorption, EPR, and resonance Raman (RR) spectroscopy of the unligated protein as well as forms in which the heme is ligated by CO or NO. To probe the oligomerization state of RCCP and its potential influence on heme reactivity, we have compared the properties of wild-type RCCP with a mutant (K42E) that lacks a salt bridge at the subunit interface. Analytical ultracentrifugation indicates that wild-type and K42E proteins are both monomeric in solution, contrary to the homodimeric structure of the crystalline state. Surprisingly, the K42E mutation produces a number of changes at the heme center (nearly 20 A distant), including perturbation of the ferric spin-state equilibrium and a change in the ferrous heme-nitrosyl complex from a six-coordinate/five-coordinate mixture to a predominantly five-coordinate heme-NO species. RR spectra indicate that ferrous K42E and wild-type RCCP both have relatively high Fe-His stretching frequencies, suggesting that the more favored five-coordinate heme-nitrosyl formation in K42E is not caused by a weaker Fe2+-His bond. Nevertheless, the altered reactivity of ferrous K42E with NO, together with its modified ferric spin state, shows that structural changes originating at the dimer interface can affect the properties of the heme center, raising the exciting possibility that intermolecular encounters at the protein surface might modulate the reactivity of cytochrome c' in vivo.

Copper-mediated dimerization of CopZ, a predicted copper chaperone from Bacillus subtilis.

Understanding the metal-binding properties and solution states of metallo-chaperones is a key step in understanding how they function in metal ion transfer. Using spectroscopic, bioanalytical and biochemical methods, we have investigated the copper-binding properties and association states of the putative copper chaperone of Bacillus subtilis, CopZ, and a variant of the protein lacking the two cysteine residues of the MXCXXC copper-binding motif. We show that copper-free CopZ exists as a monomer, but that addition of copper(I) causes the protein to associate into homodimers. The nature of the copper(I)-CopZ complex is dependent on the level of copper loading, and we report the detection of three distinct forms, containing 0.5, 1.0 and 1.5 copper(I) ions per protein. The presence of excess dithiothreitol has a significant effect on copper(I) binding to CopZ, such that, in its presence, copper(I)-CopZ occurs mainly as a monomer species. Data for copper binding to the double-cysteine variant of CopZ are consistent with an essential role for these residues in tight copper binding in the wild-type protein. We conclude that the complex nature of copper(I) binding to CopZ may underpin mechanisms of protein-to-protein copper(I) transfer.