Roberto K. Salinas

Plasticity in protein–DNA recognition: lac repressor interacts with its natural operator O1 through alternative conformations of its DNA-binding domain

The lac repressor–operator system is a model system for understanding protein–DNA interactions and allosteric mechanisms in gene regulation. Despite the wealth of biochemical data provided by extensive mutations of both repressor and operator, the specific recognition mechanism of the natural lac operators by lac repressor has remained elusive. Here we present the first high‐resolution structure of a dimer of the DNA‐binding domain of lac repressor bound to its natural operator O1. The global positioning of the dimer on the operator is dramatically asymmetric, which results in a different pattern of specific contacts between the two sites. Specific recognition is accomplished by a combination of elongation and twist by 48° of the right lac subunit relative to the left one, significant rearrangement of many side chains as well as sequence‐dependent deformability of the DNA. The set of recognition mechanisms involved in the lac repressor–operator system is unique among other protein–DNA complexes and presents a nice example of the adaptability that both proteins and DNA exhibit in the context of their mutual interaction.

Bacterial killing via a type IV secretion system

Type IV secretion systems (T4SSs) are multiprotein complexes that transport effector proteins and protein-DNA complexes through bacterial membranes to the extracellular milieu or directly into the cytoplasm of other cells. Many bacteria of the family Xanthomonadaceae, which occupy diverse environmental niches, carry a T4SS with unknown function but with several characteristics that distinguishes it from other T4SSs. Here we show that the Xanthomonas citri T4SS provides these cells the capacity to kill other Gram-negative bacterial species in a contact-dependent manner. The secretion of one type IV bacterial effector protein is shown to require a conserved C-terminal domain and its bacteriolytic activity is neutralized by a cognate immunity protein whose 3D structure is similar to peptidoglycan hydrolase inhibitors. This is the first demonstration of the involvement of a T4SS in bacterial killing and points to this special class of T4SS as a mediator of both antagonistic and cooperative interbacterial interactions.

A Component of the Xanthomonadaceae Type IV Secretion System Combines a VirB7 Motif with a N0 Domain Found in Outer Membrane Transport Proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 Å X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7XAC2622) and its interaction with VirB9. NMR solution studies show that residues 27–41 of the disordered flexible N-terminal region of VirB7XAC2622 interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7XAC2622 has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7XAC2622 oligomerizes through interactions involving conserved residues in the N0 domain and residues 42–49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB7XAC2622 oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.

Order-disorder transitions govern kinetic cooperativity and allostery of monomeric human glucokinase.

Analysis of the functional dynamics of human glucokinase reveals that a slow order-disorder transition governs monomeric kinetic cooperativity in response to glucose concentrations.

Direct Evidence of Conformational Heterogeneity in Human Pancreatic Glucokinase from High-Resolution Nuclear Magnetic Resonance

High-resolution nuclear magnetic resonance is used to investigate the conformational dynamics of human glucokinase, a 52 kDa monomeric enzyme that displays kinetic cooperativity. (1)H-(15)N transverse relaxation optimized spectra of uniformly labeled glucokinase, recorded in the absence and presence of glucose, reveal significant cross-peak overlap and heterogeneous peak intensities that persist over a range of temperatures. (15)N-specific labeling of isoleucines and tryptophans, reporting on backbone and side chain dynamics, respectively, demonstrates that both unliganded and glucose-bound enzymes sample multiple conformations, although glucose stabilizes certain conformations. These results provide the first direct evidence of glucokinase conformational heterogeneity and hence shed light on the molecular basis of cooperativity.

Ca2+ Binding Alters the Interdomain Flexibility between the Two Cytoplasmic Calcium-binding Domains in the Na+/Ca2+ Exchanger

The Na+/Ca2+ exchanger (NCX) is a membrane protein, which catalyzes the counter transport of Na+ and Ca2+ ions across the plasma membrane, playing a key role in the maintenance of the intracellular Ca2+ homeostasis in various cell types. NCX consists of a transmembrane part and a large intracellular loop. The activation of the NCX transport function requires the binding of Ca2+ to two tandem C2 domains, CBD1 and CBD2, which are an integral part of the exchangers intracellular loop. Although high-resolution structures of individual CBD1 and CBD2 are available, their interdomain structure and dynamics and the atomic level mechanism of allosteric Ca2+-regulation remains unknown. Here, we use solution NMR spectroscopy to study the interdomain dynamics of CBD12, a 32 kDa construct that contains both the CBD1 and CBD2 domains connected by a short linker. Analysis of NMR residual dipolar couplings shows that CBD12 assumes on average an elongated shape both in the absence and in the presence of Ca2+. NMR 15N relaxation data of the Apo state indicate that the two domains sample a wide range of relative arrangements on the nanosecond time scale. These arrangements comprise significantly non-linear interdomain orientations. Binding of Ca2+ to CBD1 significantly restricts the interdomain flexibility, stabilizing a more rigid elongated conformation. These findings suggest a molecular mechanism for the role of CBD12 in the function of NCX.

Altered specificity in DNA binding by the lac repressor: a mutant lac headpiece that mimics the gal repressor.

Recognition of the lac operator by the lac repressor involves specific interactions between residues in the repressors recognition helix and bases in the DNA major groove. Tyr17 and Gln18, at positions 1 and 2 in the lac repressor recognition helix, can be exchanged for other amino acids to generate mutant repressors that display altered specificity. We have solved the solution structure of a protein–DNA complex of an altered‐specificity mutant lac headpiece in which Tyr17 and Gln18 were exchanged for valine and alanine, respectively, as found in the recognition helix of the gal repressor. As previously described by Lehming et al. (EMBO J. 1987, 6, 3145–3153), this altered‐specificity mutant of the lac repressor recognizes a variant lac operator that is similar to the gal operator Oe. The mutant lac headpiece showed the predicted specificity and is also able to mimic the gal repressor by recognizing and bending the natural gal operator Oe. These structural data show that, while most of the anchoring points that help the lac headpiece to assemble on the lac operator were preserved, a different network of protein–DNA interactions connecting Ala17 and Val18 to bases in the DNA major groove drives the specificity towards the altered operator.

Solution structure of the C-terminal domain of multiprotein bridging factor 1 (MBF1) of Trichoderma reesei.

Solution structure of the C-terminal domain of multiprotein bridging factor 1 (MBF1) of Trichoderma reesei Roberto K. Salinas,* Cesar M. Camilo, Simona Tomaselli, Estela Y. Valencia, Chuck S. Farah, Hamza El-Dorry, and Felipe S. Chambergo* 1 Institute of Chemistry, University of São Paulo, São Paulo SP 05508-900, Brazil 2 Laboratorio NMR, ISMAC, CNR, Milano 20133, Italy 3 Department of Biology and the Science and Research Technology Center, The American University in Cairo, Cairo, Egypt 4 School of Arts, Sciences and Humanities, University of São Paulo, São Paulo SP 03828-000, Brazil

Deciphering the role of the electrostatic interactions in the α‐tropomyosin head‐to‐tail complex

Skeletal α‐tropomyosin (Tm) is a dimeric coiled‐coil protein that forms linear assemblies under low ionic strength conditions in vitro through head‐to‐tail interactions. A previously published NMR structure of the Tm head‐to‐tail complex revealed that it is formed by the insertion of the N‐terminal coiled‐coil of one molecule into a cleft formed by the separation of the helices at the C‐terminus of a second molecule. To evaluate the contribution of charged residues to complex stability, we employed single and double‐mutant Tm fragments in which specific charged residues were changed to alanine in head‐to‐tail binding assays, and the effects of the mutations were analyzed by thermodynamic double‐mutant cycles and protein–protein docking. The results show that residues K5, K7, and D280 are essential to the stability of the complex. Though D2, K6, D275, and H276 are exposed to the solvent and do not participate in intermolecular contacts in the NMR structure, they may contribute to head‐to‐tail complex stability by modulating the stability of the helices at the Tm termini. Proteins 2008.

VirB7 and VirB9 Interactions Are Required for the Assembly and Antibacterial Activity of a Type IV Secretion System

The type IV secretion system (T4SS) from the phytopathogen Xanthomonas citri (Xac) is a bactericidal nanomachine. The T4SS core complex is a ring composed of multiple copies of VirB7-VirB9-VirB10 subunits. Xac-VirB7 contains a disordered N-terminal tail (VirB7NT) that recognizes VirB9, and a C-terminal domain (VirB7CT) involved in VirB7 self-association. Here, we show that VirB7NT forms a short β strand upon binding to VirB9 and stabilizes it. A tight interaction between them is essential for T4SS assembly and antibacterial activity. Abolishing VirB7 self-association or deletion of the VirB7 C-terminal domain impairs this antibacterial activity without disturbing T4SS assembly. These findings reveal protein interactions within the core complex that are critical for the stability and activity of a T4SS.