NMR detection of slow conformational dynamics in an endonuclease toxin

The cytotoxic activity of the secreted bacterial toxin colicin E9 is due to a non-specific DNase housed in the C-terminus of the protein. Double-resonance and triple-resonance NMR studies of the 134-amino acid¹⁵ N- and ¹³C/¹⁵N-labelled DNase domain are presented. Extensive conformational heterogeneity was evident from the presence of far more resonances than expected based on the amino acid sequence of the DNase, and from the appearance of chemical exchange cross-peaks in TOCSY and NOESY spectra. EXSY spectra were recorded to confirm that slow chemical exchange was occurring. Unambiguous sequence-specific resonance assignments are presented for one region of the protein, Pro⁶⁵-Asn⁷², which exists in two slowly exchanging conformers based on the identification of chemical exchange cross-peaks in 3D ¹H-¹H-¹⁵N EXSY-HSQC, NOESY-HSQC and TOCSY-HSQC spectra, together with C and C chemical shifts measured in triple-resonance spectra and sequential NH NOEs. The rates of conformational exchange for backbone amide resonances in this stretch of amino acids, and for the indole NH of either Trp²² or Trp⁵⁸, were determined from the intensity variation of the appropriate diagonal and chemical exchange cross-peaks recorded in 3D¹ H-¹H-¹⁵N NOESY-HSQC spectra. The data fitted a model in which this region of the DNase has two conformers, N_A and N_B, which interchange at 15 °C with a forward rate constant of 1.61 ± 0.5 s^-1 and a backward rate constant of 1.05 ± 0.5 s^-1. Demonstration of this conformational equilibrium has led to a reappraisal of a previously proposed kinetic scheme describing the interaction of E9 DNase with immunity proteins [Wallis et al. (1995) Biochemistry, 34, 13743–13750 and 13751–13759]. The revised scheme is consistent with the specific inhibitor protein for the E9 DNase, Im9, associating with both the N_A and N_B conformers of the DNase and with binding only to the N_B conformer detected because the rate of dissociation of the complex of Im9 and the N_A conformer, N_AI, is extremely rapid. In this model stoichiometric amounts of Im9 convert, the E9 DNase is converted wholly into the N_BI form. The possibility that cis–trans isomerisation of peptide bonds preceding proline residues is the cause of the conformational heterogeneity is discussed. E9 DNase contains 10 prolines, with two bracketing the stretch of amino acids that have allowed the N_A N_B interconversion to be identified, Pro⁶⁵ and Pro⁷³. The model assumes that one or both of these can exist in either the cis or trans form with strong Im9 binding possible to only one form.     

Identification of a cysteine residue at the active site of Escherichia coli isocitrate lyase.

Escherichia coli isocitrate lyase was inactivated by iodacetate in a pseudo-first-order process. Complete inactivation was associated with the incorporation of only one carboxymethyl group per enzyme subunit. The substrate and products of the enzyme protected against inactivation, suggesting that the reactive group may be located at the active site. Isolation and sequencing of a carboxymethylated peptide showed that the modified residue was a cysteine, in the sequence Cys-Gly-His-Met-Gly-Gly-Lys. The reactivity of isocitrate lyase to iodoacetate declined with pH, following a titration curve for a group of pKa 7.1. The Km of the enzyme for isocritrate declined over the same pH range.     

Colicin Biology

Colicins are proteins produced by and toxic for some strains of Escherichia coli. They are produced by strains of E. coli carrying a colicinogenic plasmid that bears the genetic determinants for colicin synthesis, immunity, and release. Insights gained into each fundamental aspect of their biology are presented: their synthesis, which is under SOS regulation; their release into the extracellular medium, which involves the colicin lysis protein; and their uptake mechanisms and modes of action. Colicins are organized into three domains, each one involved in a different step of the process of killing sensitive bacteria. The structures of some colicins are known at the atomic level and are discussed. Colicins exert their lethal action by first binding to specific receptors, which are outer membrane proteins used for the entry of specific nutrients. They are then translocated through the outer membrane and transit through the periplasm by either the Tol or the TonB system. The components of each system are known, and their implication in the functioning of the system is described. Colicins then reach their lethal target and act either by forming a voltage-dependent channel into the inner membrane or by using their endonuclease activity on DNA, rRNA, or tRNA. The mechanisms of inhibition by specific and cognate immunity proteins are presented. Finally, the use of colicins as laboratory or biotechnological tools and their mode of evolution are discussed.     

Effect of different ankle- and knee-joint positions on gastrocnemius medialis fascicle length and EMG activity during isometric plantar flexion

The purpose of this study was to provide evidence on the fact that the observed decrease in EMG activity of the gastrocnemius medialis (GM) at pronounced knee flexed positions is not only due to GM insufficiency, by examining muscle fascicle lengths during maximal voluntary contractions at different positions. Twenty-two male long distance runners (body mass: 78.5+/-6.7 kg, height: 183+/-6 cm) participated in the study. The subjects performed isometric maximal voluntary plantar flexion contractions (MVC) of their left leg at six ankle-knee angle combinations. To examine the resultant ankle joint moments the kinematics of the left leg were recorded using a Vicon 624 system with 8 cameras operating at 120 Hz. The EMG activity of GM, gastrocnemius lateralis (GL), soleus (SOL) and tibialis anterior (TA) were measured using surface electromyography. Synchronously, fascicle length and pennation angle values of the GM were obtained at rest and at the plateau of the maximal plantar flexion using ultrasonography. The main findings were: (a) identifiable differences in fascicle length of the GM at rest do not necessarily imply that these differences would also exist during a maximal isometric plantar flexion contraction and (b) the EMG activity of the biarticular GM during the MVC decreased at a pronounced flexed knee-joint position (up to 110 degrees ) despite of no differences in GM fascicle length. It is suggested that the decrease in EMG activity of the GM at pronounced knee flexed positions is due to a critical force-length potential of all three muscles of the triceps surae.     

Galactose recognition by the apicomplexan parasite Toxoplasma gondii

Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system.     

Kinetic basis for the competitive recruitment of TolB by the intrinsically disordered translocation domain of colicin E9

TolB and Pal are members of the Tol-Pal system that spans the cell envelope of Gram-negative bacteria and contributes to the stability and integrity of the bacterial outer membrane (OM). Lipoylated Pal is tethered to the OM and binds the β-propeller domain of periplasmic TolB, which, as recent evidence suggests, disengages TolB from its interaction with other components of the Tol system in the inner membrane. Antibacterial nuclease colicins such as colicin E9 (ColE9) also bind the β-propeller domain of TolB in order to catalyze their translocation across the bacterial OM. In contrast to Pal, however, colicin binding to TolB promotes its interaction with other components of the Tol system. Here, through a series of pre-steady-state kinetic experiments utilizing fluorescence resonance energy transfer pairs within the individual protein-protein complexes, we establish the kinetic basis for such 'competitive recruitment' by the TolB-binding epitope (TBE) of ColE9. Surprisingly, the 16-residue disordered ColE9 TBE associates more rapidly with TolB than Pal, a folded 13-kDa protein. Moreover, we demonstrate that calcium ions, which bind within the confines of the TolB β-propeller domain tunnel and are known to increase the affinity of the TolB-ColE9 complex, do not exert their influence through long-range electrostatic effects, as had been predicted, but through short-range effects that slow the dissociation rate of ColE9 TBE from its complex with TolB. Our study demonstrates that an intrinsically disordered protein undergoing binding-induced folding can compete effectively with a globular protein for a common target by associating more rapidly than the globular protein.     

Experimental and computational analyses of the energetic basis for dual recognition of immunity proteins by colicin endonucleases

Colicin endonucleases (DNases) are bound and inactivated by immunity (Im) proteins. Im proteins are broadly cross-reactive yet specific inhibitors binding cognate and non-cognate DNases with K_d values that vary between 10^− 4 and 10^− 14 M, characteristics that are explained by a ‘dual-recognition’ mechanism. In this work, we addressed for the first time the energetics of Im protein recognition by colicin DNases through a combination of E9 DNase alanine scanning and double-mutant cycles (DMCs) coupled with kinetic and calorimetric analyses of cognate Im9 and non-cognate Im2 binding, as well as computational analysis of alanine scanning and DMC data. We show that differential ΔΔGs observed for four E9 DNase residues cumulatively distinguish cognate Im9 association from non-cognate Im2 association. E9 DNase Phe86 is the primary specificity hotspot residue in the centre of the interface, which is coordinated by conserved and variable hotspot residues of the cognate Im protein. Experimental DMC analysis reveals that only modest coupling energies to Im9 residues are observed, in agreement with calculated DMCs using the program ROSETTA and consistent with the largely hydrophobic nature of E9 DNase-Im9 specificity contacts. Computed values for the 12 E9 DNase alanine mutants showed reasonable agreement with experimental ΔΔG data, particularly for interactions not mediated by interfacial water molecules. ΔΔG predictions for residues that contact buried water molecules calculated using solvated rotamer models met with mixed success; however, we were able to predict with a high degree of accuracy the location and energetic contribution of one such contact. Our study highlights how colicin DNases are able to utilise both conserved and variable amino acids to distinguish cognate from non-cognate Im proteins, with the energetic contributions of the conserved residues modulated by neighbouring specificity sites.     

Catalytic cycle of human glutathione reductase near 1 A resolution

Efficient enzyme catalysis depends on exquisite details of structure beyond those resolvable in typical medium- and high-resolution crystallographic analyses. Here we report synchrotron-based cryocrystallographic studies of natural substrate complexes of the flavoenzyme human glutathione reductase (GR) at nominal resolutions between 1.1 and 0.95 Å that reveal new aspects of its mechanism. Compression in the active site causes overlapping van der Waals radii and distortion in the nicotinamide ring of the NADPH substrate, which enhances catalysis via stereoelectronic effects. The bound NADPH and redox-active disulfide are positioned optimally on opposite sides of the flavin for a 1,2-addition across a flavin double bond. The new structures extend earlier observations to reveal that the redox-active disulfide loop in GR is an extreme case of sequential peptide bonds systematically deviating from planarity—a net deviation of 53° across five residues. But this apparent strain is not a factor in catalysis, as it is present in both oxidized and reduced structures. Intriguingly, the flavin bond lengths in oxidized GR are intermediate between those expected for oxidized and reduced flavin, but we present evidence that this may not be due to the protein environment but instead due to partial synchrotron reduction of the flavin by the synchrotron beam. Finally, of more general relevance, we present evidence that the structures of synchrotron-reduced disulfide bonds cannot generally be used as reliable models for naturally reduced disulfide bonds.