Histidine kinases (HKs) are major players in bacterial signaling. There has been an explosion of new HK crystal structures in the last five years. We globally analyze the structures of HKs to yield insights into the mechanisms by which signals are transmitted to and across protein structures in this family. We interpret known enzymological data in the context of new structural data to show how asymmetry across the dimer interface is a key feature of signal transduction in HKs, and discuss how different HK domains undergo asymmetric-to-symmetric transitions during signal transduction and catalysis. A thermodynamic framework for signaling that encompasses these various properties is presented and the consequences of weak thermodynamic coupling are discussed. The synthesis of observations from enzymology, structural biology, protein engineering and thermodynamics paves the way for a deeper molecular understanding of histidine kinase signal transduction.

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An uclear receptor, peroxisome proliferator-activated receptor g (PPARg ), is al igand-dependent transcription factor involved in glucose homeostasis anda dipocyted ifferentiation. PPARg is the molecular target of various natural ands ynthetic molecules, includinga nti-diabetic agents such as rosiglitazone. Amide hydrogen/deuterium-exchange (H/D-Ex), coupled with proteolysis and mass spectrometry,w as applied to study the dynamics of theP PAR g ligand binding domain (LBD) with or withoutmolecules that modulate PPARg activity.The H/D-Ex patterns of ligand-free PPARg LBD show that the ligand binding pocket of LBD is significantly more dynamic than ther est of the LBD. Presumably,t he bindingp ocketi si ntrinsically disordered in order to accommodated ifferentl igands. The presence of twof ull agonists (rosiglitazone and GW1929), ap artial agonist (nTZDpa), and ac ovalenta ntagonist (GW9662), changedt he dynamics/conformation of PPARg LBD ands lowedt he H/D exchanger ate in variousr egionso ft he protein.T he full agonists slowed theH /D exchange more globally andtoagreater extent than the partial agonist or the antagonist, indicating thatthe full agonist stabilizes the PPARg LBDmorethan thepartial agonist or the antagonist. Oneinteresting observation is that thetwo fullagonists significantly stabilized helix12whilethe partial agonist andthe antagonist did not perturb the H/D exchange of this region. Ther esults showedt hatt he change in proteind ynamics induced by ligandb inding may be an important factor fort he activation of genes and that H/D-Ex is au sefulm ethodf or analyzingt he biological activity of drug leads.

Statistical analysis of data from 39 proteins (13 766 amino acid residues) digested with immobilized porcine pepsin under conditions compatible with hydrogen/deuterium (H/D) exchange (<1 degrees C, <30 s) was performed to examine pepsin cleavage specificity. The cleavage of pepsin was most influenced by the amino acid residue at position P1. Phe and Leu are favored residues each with a cleavage probability greater than 40%. His, Lys, Arg, or Pro residues prohibit cleavage when found at the P1 position. Pro also cannot be at position P2 (cleavage probability <0.3%). Occupation of the P3 position by His, Lys, or Arg, or occupation of the P2' position by Pro, also leads to very little cleavage (cleavage probability <1.7%). The average cleavage probability over the entire data set was 13.6%, which is slightly lower than the value previously obtained by Powers et al. (14.8%). This is due, in part, to the larger protein sizes used in the current study. While the specificity of pepsin was similar to that previously observed, higher selectivity was observed in the present study due to less experimental variation in the conditions used to generate our database.

Summary Bacteria transduce signals across the membrane using two-component systems (TCSs), consisting of a membrane-spanning sensor histidine kinase and a cytoplasmic response regulator. In Gram negative bacteria, the PhoPQ TCS senses cations and antimicrobial peptides, yet little is known about the structural changes involved in transmembrane signaling. We construct a model of PhoQ signal transduction using Bayesian inference, based on disulfide crosslinking data and homologous crystal structures. The data are incompatible with a single conformation but are instead consistent with two interconverting structures. These states differ in membrane depth of the periplasmic acidic patch and the reciprocal displacement of diagonal helices along the dimer interface. Studies of multiple histidine kinases suggest this repacking might be a common mode of signal transduction in sensor His-kinase receptors. Since a similar scissors model has been ruled out in CheA-linked chemoreceptors, the new evidence suggests that sensor His-kinase and CheA-linked receptors possess different signaling mechanisms.

Ion-exchange (IEX) chromatography steps are widely applied in protein purification processes because of their high capacity, selectivity, robust operation, and well-understood principles. Optimization of IEX steps typically involves resin screening and selection of the pH and counterion concentrations of the load, wash, and elution steps. Time and material constraints associated with operating laboratory columns often preclude evaluating more than 20-50 conditions during early stages of process development. To overcome this limitation, a high-throughput screening (HTS) system employing a robotic liquid handling system and 96-well filterplates was used to evaluate various operating conditions for IEX steps for monoclonal antibody (mAb) purification. A screening study for an adsorptive cation-exchange step evaluated eight different resins. Sodium chloride concentrations defining the operating boundaries of product binding and elution were established at four different pH levels for each resin. Adsorption isotherms were measured for 24 different pH and salt combinations for a single resin. An anion-exchange flowthrough step was then examined, generating data on mAb adsorption for 48 different combinations of pH and counterion concentration for three different resins. The mAb partition coefficients were calculated and used to estimate the characteristic charge of the resin-protein interaction. Host cell protein and residual Protein A impurity levels were also measured, providing information on selectivity within this operating window. The HTS system shows promise for accelerating process development of IEX steps, enabling rapid acquisition of large datasets addressing the performance of the chromatography step under many different operating conditions.

The integrin αIIbβ3 is a transmembrane (TM) heterodimeric adhesion receptor that exists in equilibrium between resting and active ligand binding conformations. In resting αIIbβ3, the TM and cytoplasmic domains of αIIb and β3 form a heterodimer that constrains αIIbβ3 in its resting conformation. To study the structure and dynamics of the cytoplasmic domain heterodimer, we prepared a disulfide-stabilized complex consisting of portions of the TM domains and the full cytoplasmic domains. NMR and hydrogendeuterium exchange of this complex in micelles showed that the αIIb cytoplasmic domain is largely disordered, but it interacts with and influences the conformation of the β3 cytoplasmic domain. The β3 cytoplasmic domain consists of a stable proximal helix contiguous with the TM helix and two distal amphiphilic helices. To confirm the NMR structure in a membrane-like environment, we studied the β3 cytoplasmic domain tethered to phospholipid bilayers. Hydrogen-deuterium exchange mass spectrometry, as well as circular dichroism spectroscopy, demonstrated that the β3 cytoplasmic domain becomes more ordered and helical under these conditions, consistent with our NMR results. Further, these experiments suggest that the two distal helices associate with lipid bilayers but undergo fluctuations that would allow rapid binding of cytoplasmic proteins regulating integrin activation, such as talin and kindlin-3. Thus, these results provide a framework for understanding the kinetics and thermodynamics of protein interactions involving integrin cytoplasmic domains and suggest that such interactions act in a concerted fashion to influence integrin stalk separation and exposure of extracellular ligand binding sites.platelets | fibrinogen receptors | receptor regulation

At least 119 mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis by an unidentified toxic gain of function. We compared the dynamic properties of 13 as-isolated, partially metallated, SOD1 variant enzymes using hydrogen-deuterium exchange. We identified a shared property of these familial amyotrophic lateral sclerosis-related SOD1 variants, namely structural and dynamic change affecting the electrostatic loop (loop VII) of SOD1. Furthermore, SOD1 variants that have severely compromised metal binding affinities demonstrated additional structural and dynamic changes to the zinc-binding loop (loop IV) of SOD1. Although the biological consequences of increased loop VII mobility are not fully understood, this common property is consistent with the hypotheses that SOD1 mutations exert toxicity via aggregation or aberrant association with other cellular constituents.At least 119 mutations in the gene encoding copper/zinc superoxide dismutase cause fALS 3 by introducing an unknown toxic gain of function. Numerous hypotheses have been proposed to explain mechanisms of SOD1 variant toxicity (reviewed in Refs. 2 and 3). Proposed hypotheses to define the structural or functional alterations shared by SOD1 variants include: 1) decreased stability of apo (metal-deficient) or metallated SOD1 (4), 2) increased hydrophobicity (5), 3) increased self-aggregation propensity (6), 4) increased susceptibility to post-translational modification, 5) decreased metal affinity (7), 6) destabilization of the SOD1 native dimer, and 7) aberrant copper-mediated chemistry (8). The literature, as a whole, supports the notion that the aforementioned hypotheses are in fact related, because SOD1 metal content, native disulfide bond status, hydrophobicity, and stability are interrelated (2) and may thereby affect the specificity of copper-mediated chemistry (9) and aggregation propensity.Proposed hypotheses to explain the biological consequences of SOD1 toxicity include: 1) impairment of axonal transport (10), 2) excitotoxicity (11), 3) impairment of proteasome (12) or chaperone activity (13), and 4) mitochondrial (14, 15) or endoplasmic reticulum-Golgi dysfunction (15). The relationship of mutation-associated structural changes of SOD1 with these downstream effects is not well established. SOD1 aggregation has been implicated as a contributor to mitochondrial (15, 16), endoplasmic reticulum-Golgi (15, 17, 18), proteasome (19), and chaperone (13) dysfunction and thus bridges the conceptual gap between mutation-related changes to the physicochemical properties of SOD1 and the varied cellular consequences of SOD1 mutations. Indeed, the only property shared by all fALS SOD1 variants thus far studied is an increased propensity to form proteinaceous aggregates of SOD1, as evidenced in fALS patients (20, 21), 21 rodent lines of 13 different SOD1 mutations (22), and at least 13 fALS mutations in cell culture models. Moreover, proteinaceous SOD1-containing inclusions were also found in a subset of spora...