Structure-Based Predictive Models for Allosteric Hot Spots

In allostery, a binding event at one site in a protein modulates the behavior of a distant site. Identifying residues that relay the signal between sites remains a challenge. We have developed predictive models using support-vector machines, a widely used machine-learning method. The training data set consisted of residues classified as either hotspots or non-hotspots based on experimental characterization of point mutations from a diverse set of allosteric proteins. Each residue had an associated set of calculated features. Two sets of features were used, one consisting of dynamical, structural, network, and informatic measures, and another of structural measures defined by Daily and Gray [1]. The resulting models performed well on an independent data set consisting of hotspots and non-hotspots from five allosteric proteins. For the independent data set, our top 10 models using Feature Set 1 recalled 68–81% of known hotspots, and among total hotspot predictions, 58–67% were actual hotspots. Hence, these models have precision P = 58–67% and recall R = 68–81%. The corresponding models for Feature Set 2 had P = 55–59% and R = 81–92%. We combined the features from each set that produced models with optimal predictive performance. The top 10 models using this hybrid feature set had R = 73–81% and P = 64–71%, the best overall performance of any of the sets of models. Our methods identified hotspots in structural regions of known allosteric significance. Moreover, our predicted hotspots form a network of contiguous residues in the interior of the structures, in agreement with previous work. In conclusion, we have developed models that discriminate between known allosteric hotspots and non-hotspots with high accuracy and sensitivity. Moreover, the pattern of predicted hotspots corresponds to known functional motifs implicated in allostery, and is consistent with previous work describing sparse networks of allosterically important residues.     

Experiments on Hemoglobin in Single Crystals and Silica Gels Distinguish among Allosteric Models

Trapping quaternary structures of hemoglobin in single crystals or by encapsulation in silica gels has provided a demanding set of data to test statistical mechanical models of allostery. In this work, we compare the results of those experiments with predictions of the four major allosteric models for hemoglobin: the quaternary two-state model of Monod, Wyman, and Changeux; the tertiary two-state model of Henry et al., which is the simplest extension of the Monod-Wyman-Changeux model to include pre-equilibria of tertiary as well as quaternary conformations; the structure-based model of Szabo and Karplus; and the modification of the latter model by Lee and Karplus. We show that only the tertiary two-state model can provide a near quantitative explanation of the single-crystal and gel experimental results.     

Allosteric Transitions of Supramolecular Systems Explored by Network Models: Application to Chaperonin GroEL

Identification of pathways involved in the structural transitions of biomolecular systems is often complicated by the transient nature of the conformations visited across energy barriers and the multiplicity of paths accessible in the multidimensional energy landscape. This task becomes even more challenging in exploring molecular systems on the order of megadaltons. Coarse-grained models that lend themselves to analytical solutions appear to be the only possible means of approaching such cases. Motivated by the utility of elastic network models for describing the collective dynamics of biomolecular systems and by the growing theoretical and experimental evidence in support of the intrinsic accessibility of functional substates, we introduce a new method, adaptive anisotropic network model (aANM), for exploring functional transitions. Application to bacterial chaperonin GroEL and comparisons with experimental data, results from action minimization algorithm, and previous simulations support the utility of aANM as a computationally efficient, yet physically plausible, tool for unraveling potential transition pathways sampled by large complexes/assemblies. An important outcome is the assessment of the critical inter-residue interactions formed/broken near the transition state(s), most of which involve conserved residues.     

Selective Allosteric Inhibition of MMP9 Is Efficacious in Preclinical Models of Ulcerative Colitis and Colorectal Cancer

Expression of matrix metalloproteinase 9 (MMP9) is elevated in a variety of inflammatory and oncology indications, including ulcerative colitis and colorectal cancer. MMP9 is a downstream effector and an upstream mediator of pathways involved in growth and inflammation, and has long been viewed as a promising therapeutic target. However, previous efforts to target matrix metalloproteinases (MMPs), including MMP9, have utilized broad-spectrum or semi-selective inhibitors. While some of these drugs showed signs of efficacy in patients, all MMP-targeted inhibitors have been hampered by dose-limiting toxicity or insufficient clinical benefit, likely due to their lack of specificity. Here, we show that selective inhibition of MMP9 did not induce musculoskeletal syndrome (a characteristic toxicity of pan-MMP inhibitors) in a rat model, but did reduce disease severity in a dextran sodium sulfate-induced mouse model of ulcerative colitis. We also found that MMP9 inhibition decreased tumor growth and metastases incidence in a surgical orthotopic xenograft model of colorectal carcinoma, and that inhibition of either tumor- or stroma-derived MMP9 was sufficient to reduce primary tumor growth. Collectively, these data suggest that selective MMP9 inhibition is a promising therapeutic strategy for treatment of inflammatory and oncology indications in which MMP9 is upregulated and is associated with disease pathology, such as ulcerative colitis and colorectal cancer. In addition, we report the development of a potent and highly selective allosteric MMP9 inhibitor, the humanized monoclonal antibody GS-5745, which can be used to evaluate the therapeutic potential of MMP9 inhibition in patients.     

An Orally Active Allosteric GLP-1 Receptor Agonist Is Neuroprotective in Cellular and Rodent Models of Stroke

Diabetes is a major risk factor for the development of stroke. Glucagon-like peptide-1 receptor (GLP-1R) agonists have been in clinical use for the treatment of diabetes and also been reported to be neuroprotective in ischemic stroke. The quinoxaline 6,7-dichloro-2-methylsulfonyl-3-N-tert- butylaminoquinoxaline (DMB) is an agonist and allosteric modulator of the GLP-1R with the potential to increase the affinity of GLP-1 for its receptor. The aim of this study was to evaluate the neuroprotective effects of DMB on transient focal cerebral ischemia. In cultured cortical neurons, DMB activated the GLP-1R, leading to increased intracellular cAMP levels with an EC₅₀ value about 100 fold that of exendin-4. Pretreatment of neurons with DMB protected against necrotic and apoptotic cell death was induced by oxygen-glucose deprivation (OGD). The neuroprotective effects of DMB were blocked by GLP-1R knockdown with shRNA but not by GLP-1R antagonism. In C57BL/6 mice, DMB was orally administered 30 min prior to middle cerebral artery occlusion (MCAO) surgery. DMB markedly reduced the cerebral infarct size and neurological deficits caused by MCAO and reperfusion. The neuroprotective effects were mediated by activation of the GLP-1R through the cAMP-PKA-CREB signaling pathway. DMB exhibited anti-apoptotic effects by modulating Bcl-2 family members. These results provide evidence that DMB, a small molecular GLP-1R agonist, attenuates transient focal cerebral ischemia injury and inhibits neuronal apoptosis induced by MCAO. Taken together, these data suggest that DMB is a potential neuroprotective agent against cerebral ischemia.     

Allosteric transition in triply met-haemoglobin

Methaemoglobin undergoes a transition to a T-like form at acid pH in the presence of strong effectors such as inositol hexakisphosphate (IHP), as evidenced by spectroscopic and oxidation potential measurements. Since oxygen and CO do not bind to the ferric haems, it is difficult to compare the properties of the R-met and T-met forms with those of ferrous haemoglobin. We have therefore prepared 90% oxidized samples, where the dominant signal for ligand (oxygen or CO) binding is due to tetramers with three met haems. Measurements were made of the oxygen equilibrium curves and CO rebinding kinetics after photodissociation. Without effectors, the partially oxidized samples show mainly R-state properties. Addition of IHP at acid pH induces an increase in T-state behaviour, as indicated by a lower oxygen affinity and a higher fraction of the slow bimolecular component for CO rebinding.     

Allosteric movements in eubacterial RecA

The action of RecA, an important eubacterial protein involved in recombination and repair, involves the transition from an inactive filament in the absence of DNA to an active filament formed in association with DNA and ATP. The structure of the inactive filament was first established in Escherichia coli RecA (EcRecA). The interaction of RecA with non-hydrolysable ATP analogues and ADP has been thoroughly characterized and the DNA binding loops visualized based on the crystal structures of the RecA proteins from Mycobacterium tuberculosis (MtRecA) and Mycobacterium smegmatis (MsRecA). A switch residue, which triggers the transformation of the information on ATP binding to the DNA binding regions, has been identified. The 20-residue C-terminal stretch of RecA, which is disordered in all other relevant crystal structures, has been defined in an MsRecA-dATP complex. The ordering of the stretch is accompanied by the generation of a new nucleotide binding site which can communicate with the original nucleotide binding site of an adjacent molecule in the filament. The plasticity of MsRecA and its mutants involving the switch residue has been explored by studying crystals grown under different conditions at two different temperatures and, in one instance, at low humidity. The structures of these crystals and those of EcRecA and Deinococcus radiodurans RecA (DrRecA) provide information on correlated movements involving different regions of the molecule. These correlated movements appear to be important in the allosteric transitions of RecA during its action.     

Allosteric agonist modulators

This article discusses a model to describe the effects of molecules that bind to a site on the receptor separate from that of the endogenous agonist to actively produce receptor signals (direct agonism). In addition, these molecules also modify the biological responses of the endogenous agonist (either potentiation or antagonism). The effects of such compounds in high-throughput screening assays are described as well as their effects on the dose-response curves to conventional agonists.     

Allosteric interactions in phosphorylase B

An allosteric model of phosphorylase B is proposed based on the following assumptions. The enzyme consists of two sub-units and undergoes a concerted transition from the inactive T to the active R state. The binding of substrates, phosphate, and glycogen is regarded as exclusive, but the binding of the activator AMP is nonexclusive. The enzyme model is of the K, V type, i. e., the activator AMP is important, not only for the T-R transition and the substrates binding, but also for the formation of the active site. Therefore, it displays a big influence on the maximal reaction rate. Calculations based on this model lead to an equation containing 5 constants, which can be easily computed from kinetic data. All kinetic measurements fit the expressions derived from the model. Independent methods for the measurement of all the constants involved were developed. They are based on the study of binding of phosphorylase with the substrates and the activator. These measurements are in satisfactory agreement with the data obtained from enzyme kinetics.     

Allosteric disulfide bonds

Disulfide bonds have been generally considered to be either structural or catalytic. Structural bonds stabilize a protein, while catalytic bonds mediate thiol-disulfide interchange reactions in substrate proteins. There is emerging evidence for a third type of disulfide bond that can control protein function by triggering a conformational change when it breaks and/or forms. These bonds can be thought of as allosteric disulfides. To better define the properties of allosteric disulfides, we have analyzed the geometry and dihedral strain of 6874 unique disulfide bonds in 2776 X-ray structures. A total of 20 types of disulfide bonds were identified in the dataset based on the sign of the five chi angles that make up the bond. The known allosteric disulfides were all contained in 1 of the 20 groups, the -RHStaple bonds. This bond group has a high mean potential energy and narrow energy distribution, which is consistent with a functional role. We suggest that the -RHStaple configuration is a hallmark of allosteric disulfides. About 1 in 15 of all structurally determined disulfides is a potential allosteric bond.     

Allosteric Agonist Modulators

This article discusses a model to describe the effects of molecules that bind to a site on the receptor separate from that of the endogenous agonist to actively produce receptor signals (direct agonism). In addition, these molecules also modify the biological responses of the endogenous agonist (either potentiation or antagonism). The effects of such compounds in high-throughput screening assays are described as well as their effects on the dose-response curves to conventional agonists.     

The effect of redox potential on the kinetics of fluorescence induction in pea chloroplasts. II. Sigmoidicity

The shape of the fluorescence induction curve in chloroplasts inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea has been determined at different redox potentials. At ?10 mV a monophasic and sigmoidal curve is seen which is transformed into an exponential curve when the potential is poised at ?150 mV. At this potential, the quencher with high midpoint, Q_H, is reduced but that with low midpoint, Q_L, is oxidized. Thus, a sigmoidal induction is observed during photoreduction of Q_L and Q_H but photoreduction of Q_L proceeds with exponential kinetics. A correlation between the relative proportions of Q_L and Q_H observed in redox titration and the sigmoidicity of induction is also seen upon depletion of Mg²⁺ and after alkalinization to pH 9.5. Several models are discussed to explain the relationship between Photosystem II interactions and Q heterogeneity.     

Allosteric transitions in cobalt hemoglobins.

Circular dichroism and difference ultraviolet visible spectra were obtained for cobalt hemoglobin derivatives. At 287 nm the ellipticity difference between the oxy- and deoxycobaltohemoglobin is about one-half as great as that for the native proteins indicating smaller quaternary conformational changes for the former. Deoxygenation increases the Soret rotational strengths of both iron and cobalt hemoglobins to comparable degrees suggesting similar conformational changes for their aromatic residues near the "heme." Deoxygenation causes a much larger decrease of L band ellipticity for iron than cobalt hemoglobin. Circular dichroism spectra of nitrosylcobaltohemoglobin indicate the molecule to have a T quaternary structure. The circular dichroism spectra of cobaltihemoglobin do not seem to fit the patterns of the other cobalt derivatives and its 287 nm ellipticity is pH-dependent. From the shape of the Soret circular dichroism spectra, it is estimated that the transition dipole makes an angle with the line joining the two opposing pyrrole nitrogens of about 60 degrees for oxy- and deoxycobaltohemoglobin, 80 degrees for cobaltihemoglobin, as compared to 70 degrees for the native oxy- and deoxyhemoglobins. Inositol hexaphosphate has little or no effect on the circular dichroism spectra of cobalt hemoglobins in the 287 nm region, but it significantly increases the Soret rotational strength and decreases the L band ellipticity. The results are interpreted to mean that polyphosphates modify primarily the protein structure of hemoglobins at the tertiary level, and that the intersubunit interactions are weak in cobalt hemoglobins.     

Dynamics of Allosteric Transitions in Dynein

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Allosteric Properties of Acetylcholinesterase

SEVERAL reports^1–3 have suggested that cholinergic ligands bind to acetylcholinesterase at sites distinct from the active centre. Changeux et al.⁴ demonstrated that acetylcholine binds to non-catalytic sites as well as to the active centre of the enzyme. Atropine arid 1-hyoscyamine have also been shown to interact with the enzyme at a region distinct from the active site^{5, 6}. We have therefore investigated whether acetylcholine and atropine are bound to the same site and whether this site is related to the acetylcholine receptor.