Selectivity by Small-Molecule Inhibitors of Protein Interactions Can Be Driven by Protein Surface Fluctuations

Small-molecules that inhibit interactions between specific pairs of proteins have long represented a promising avenue for therapeutic intervention in a variety of settings. Structural studies have shown that in many cases, the inhibitor-bound protein adopts a conformation that is distinct from its unbound and its protein-bound conformations. This plasticity of the protein surface presents a major challenge in predicting which members of a protein family will be inhibited by a given ligand. Here, we use biased simulations of Bcl-2-family proteins to generate ensembles of low-energy conformations that contain surface pockets suitable for small molecule binding. We find that the resulting conformational ensembles include surface pockets that mimic those observed in inhibitor-bound crystal structures. Next, we find that the ensembles generated using different members of this protein family are overlapping but distinct, and that the activity of a given compound against a particular family member (ligand selectivity) can be predicted from whether the corresponding ensemble samples a complementary surface pocket. Finally, we find that each ensemble includes certain surface pockets that are not shared by any other family member: while no inhibitors have yet been identified to take advantage of these pockets, we expect that chemical scaffolds complementing these “distinct” pockets will prove highly selective for their targets. The opportunity to achieve target selectivity within a protein family by exploiting differences in surface fluctuations represents a new paradigm that may facilitate design of family-selective small-molecule inhibitors of protein-protein interactions.     

Canine Distemper Virus Envelope Protein Interactions Modulated by Hydrophobic Residues in the Fusion Protein Globular Head

Membrane fusion for morbillivirus cell entry relies on critical interactions between the viral fusion (F) and attachment (H) envelope glycoproteins. Through extensive mutagenesis of an F cavity recently proposed to contribute to F''s interaction with the H protein, we identified two neighboring hydrophobic residues responsible for severe F-to-H binding and fusion-triggering deficiencies when they were mutated in combination. Since both residues reside on one side of the F cavity, the data suggest that H binds the F globular head domain sideways.     

Molecular Simulations of a Dynamic Protein Complex: Role of Salt-Bridges and Polar Interactions in Configurational Transitions

Ion charge pairs and hydrogen bonds have been extensively studied for their roles in stabilizing protein complexes and in steering the process of protein association. Recently, it has become clear that some protein complexes are dynamic in that they interconvert between several alternate configurations. We have previously characterized one such system: the EphA2:SHIP2 SAM-SAM heterodimer by solution NMR. Here we carried out extensive all-atom molecular-dynamics simulations on a microsecond time-scale starting with different NMR-derived structures for the complex. Transitions are observed between several discernible configurations at average time intervals of 50–100 ns. The domains reorient relative to one another by substantial rotation and a slight shifting of the interfaces. Bifurcated and intermediary salt-bridge and hydrogen-bond interactions play a role in the transitions in a process that can be described as moving along a “monkey-bar”. We notice an increased density of salt bridges near protein interaction surfaces that appear to enable these transitions, also suggesting why the trajectories can become kinetically hindered in regions where fewer of such interactions are possible. In this context, even microsecond molecular-dynamics simulations are not sufficient to sample the energy landscape unless the structures remain close to their experimentally derived low-energy configurations.     

Molecular Simulations of a Dynamic Protein Complex: Role of Salt-Bridges and Polar Interactions in Configurational Transitions

Ion charge pairs and hydrogen bonds have been extensively studied for their roles in stabilizing protein complexes and in steering the process of protein association. Recently, it has become clear that some protein complexes are dynamic in that they interconvert between several alternate configurations. We have previously characterized one such system: the EphA2:SHIP2 SAM-SAM heterodimer by solution NMR. Here we carried out extensive all-atom molecular-dynamics simulations on a microsecond time-scale starting with different NMR-derived structures for the complex. Transitions are observed between several discernible configurations at average time intervals of 50–100 ns. The domains reorient relative to one another by substantial rotation and a slight shifting of the interfaces. Bifurcated and intermediary salt-bridge and hydrogen-bond interactions play a role in the transitions in a process that can be described as moving along a “monkey-bar”. We notice an increased density of salt bridges near protein interaction surfaces that appear to enable these transitions, also suggesting why the trajectories can become kinetically hindered in regions where fewer of such interactions are possible. In this context, even microsecond molecular-dynamics simulations are not sufficient to sample the energy landscape unless the structures remain close to their experimentally derived low-energy configurations.     

Consequences of Domain Insertion on the Stability and Folding Mechanism of a Protein

SlyD, the sensitive-to-lysis protein from Escherichia coli, consists of two domains. They are not arranged successively along the protein chain, but one domain, the “insert-in-flap” (IF) domain, is inserted internally as a guest into a surface loop of the host domain, which is a prolyl isomerase of the FK506 binding protein (FKBP) type. We used SlyD as a model to elucidate how such a domain insertion affects the stability and folding mechanism of the host and the guest domain. For these studies, the two-domain protein was compared with a single-domain variant SlyDΔIF, SlyD* without the chaperone domain (residues 1-69 and 130-165) in which the IF domain was removed and replaced by a short loop, as present in human FKBP12. Equilibrium unfolding and folding kinetics followed an apparent two-state mechanism in the absence and in the presence of the IF domain. The inserted domain decreased, however, the stability of the host domain in the transition region and decelerated its refolding reaction by about 10-fold. This originates from the interruption of the chain connectivity by the IF domain and its inherent instability. To monitor folding processes in this domain selectively, a Trp residue was introduced as fluorescent probe. Kinetic double-mixing experiments revealed that, in intact SlyD, the IF domain folds and unfolds about 1000-fold more rapidly than the FKBP domain, and that it is strongly stabilized when linked with the folded FKBP domain. The unfolding limbs of the kinetic chevrons of SlyD show a strong downward curvature. This deviation from linearity is not caused by a transition-state movement, as often assumed, but by the accumulation of a silent unfolding intermediate at high denaturant concentrations. In this kinetic intermediate, the FKBP domain is still folded, whereas the IF domain is already unfolded.     

Hydrophobic Substitution of Surface Residues Affects Lipase Stability in Organic Solvents

A novel lipase has been recently isolated from a local Pseudomonas sp. (GQ243724). In the present study, we have tried to increase the organic solvent stability of this lipase using site-directed mutagenesis. Eight variants N219L, N219I, N219P, N219A, N219R, N219D, S251L, and S251K were designed to change the surface hydrophobicity of this enzyme with respect to the wild-type. Among these variants, the stability of N219L and N219I significantly increased in the presence of all tested organic solvents, whereas two mutants (N219R and N219D) significantly exhibited decreased stabilities in all the organic solvent studied, suggesting that improvement of hydrophobic patches on the enzyme surface enhances the stability in organic media. Furthermore, replacing Ser²⁵¹ with hydrophobic residues on the enzyme surface dramatically diminished its stability in the tested condition. In spite of the distance of the mutated sites from the active site, the values of k _cat and K _m were affected. Finally, structural analysis of the wild-type and mutated variants was carried out in the presence and absence of some organic solvents using circular dichroism and fluorescence spectroscopy.     

Effect of Macromolecular Crowding on Protein Binding Stability: Modest Stabilization and Significant Biological Consequences

Macromolecular crowding has long been known to significantly affect protein oligomerization, and yet no direct quantitative measurements appear to have been made of its effects on the binding free energy of the elemental step of adding a single subunit. Here, we report the effects of two crowding agents on the binding free energy of two subunits in the Escherichia coli polymerase III holoenzyme. The crowding agents are found, paradoxically, to have only a modest stabilizing effect, of the order of 1 kcal/mol, on the binding of the two subunits. Systematic variations in the level of stabilization with crowder size are nevertheless observed. The data are consistent with theoretical predictions based on atomistic modeling of excluded-volume interactions with crowders. We reconcile the apparent paradox presented by our data by noting that the modest effects of crowding on elemental binding steps are cumulative, and thus lead to substantial stabilization of higher oligomers. Correspondingly, the effects of small variations in the level of crowding during the lifetime of a cell may be magnified, suggesting that crowding may play a role in increased susceptibility to protein aggregation-related diseases with aging.     

Residues in the Polar Loop of Subunit c in Escherichia coli ATP Synthase Function in Gating Proton Transport to the Cytoplasm

Rotary catalysis in F₁F₀ ATP synthase is powered by proton translocation through the membrane-embedded F₀ sector. Proton binding and release occur in the middle of the membrane at Asp-61 on the second transmembrane helix (TMH) of subunit c, which folds in a hairpin-like structure with two TMHs. Previously, the aqueous accessibility of Cys substitutions in the transmembrane regions of subunit c was probed by testing the inhibitory effects of Ag⁺ or Cd²⁺ on function, which revealed extensive aqueous access in the region around Asp-61 and on the half of TMH2 extending to the cytoplasm. In the current study, we surveyed the Ag⁺ and Cd²⁺ sensitivity of Cys substitutions in the loop of the helical hairpin and used a variety of assays to categorize the mechanisms by which Ag⁺ or Cd²⁺ chelation with the Cys thiolates caused inhibition. We identified two distinct metal-sensitive regions in the cytoplasmic loop where function was inhibited by different mechanisms. Metal binding to Cys substitutions in the N-terminal half of the loop resulted in an uncoupling of F₁ from F₀ with release of F₁ from the membrane. In contrast, substitutions in the C-terminal half of the loop retained membrane-bound F₁ after metal treatment. In several of these cases, inhibition was shown to be due to blockage of passive H⁺ translocation through F₀ as assayed with F₀ reconstituted into liposomes. The results suggest that the C-terminal domain of the cytoplasmic loop may function in gating H⁺ translocation to the cytoplasm.     

Cell Membrane Stability and Leaf Surface Wax Content as Affected by Increasing Water Deficits in Maize

A field experiment was conducted with a water-stressed treatmentand well-watered control using eight maize (Zea mays L.) cultivars.Effects of water deficits on cell membrane stability (CMS) measuredby the polyethylene glycol (PEG) test, leaf surface wax content,and relative growth rate were investigated. Cytoplasmic lipidcontent was also analysed. Cell membrane stability and leaf surface wax content increasedwith the degrees of stress. Tolerance to drought evaluated asincrease in CMS under water deficit conditions was well differentiatedbetween cultivars and was well correlated with a reduction inrelative growth rate under stress. A negative correlation wasfound between percentage injury in the PEG test and leaf surfacewax content. High phospholipid contents were observed in tissuesof drought tolerant cultivars under water deficit conditions. Key words: Cell membrane stability, cytoplasmic lipid, drought tolerance, leaf surface wax, relative growth rate     

RNA结合蛋白质HuR通过增加YAP1 mRNA稳定性促进白血病细胞增殖

RNA结合蛋白质人类抗原R(Hu R)与多种实体肿瘤的发生发展密切相关,但其在人急性髓系白血病(acutemyeloidleukemia,AML)中的功能和分子机制仍未阐明。本研究收集了30例临床初诊的AML病人和30例正常对照的外周血,分离单个核细胞,进行RT-qPCR法检测。结果显示,与正常对照相比,HuR在AML病人中呈显著表达上调趋势(3.17±1.12,P<0.05)。在AML细胞系HL-60中的功能检测发现,敲低内源性HuR后,对HL-60细胞培养12 h(0.17±0.07),24 h(0.38±0.05),36 h(0.51±0.03),48 h(0.69±0.05),60 h(0.92±0.08)和72 h(1.04±0.10)的增殖产生抑制作用(P<0.05)。同时,阻滞在G1期(47.3%±5.4)的HL-60细胞比例显著升高(P<0.05),而S期(37.5%±6.9)细胞比例显著降低(P<0.05)。相反,HuR的表达抑制对HL-60细胞向单核系分化产生促进作用。RNA免疫共沉淀法检测发现,HuR可与Hippo通路的Yes相关蛋白1关键效应基因(YAP1)的mRNA结合。后续的RNA稳定性检测发现,HuR的结合可以增强YAP1 mRNA的稳定性,进而促进YAP1的表达,并进一步影响YAP1下游基因的转录。综上所述,RNA结合蛋白质HuR可通过促进Hippo通路YAP1的表达参与AML发生发展的调节。     

Reconstitution and Engineering of Apoptotic Protein Interactions on the Bacterial Cell Surface

The interactions between pro- and anti-apoptotic members of the Bcl-2 class of proteins control whether a cell lives or dies, and the study of these protein-protein interactions has been an area of intense research. In this report, we describe a new tool for the study and engineering of apoptotic protein interactions that is based on the flow cytometric detection of these interactions on the surface of Escherichia coli. After validation of the assay with the well-studied interaction between the Bak(72-87) peptide and the anti-apoptotic protein Bcl-x_L, the effect of both increasing and decreasing Bak peptide length on Bcl-x_L binding was investigated. Previous work demonstrated that the Bak(72-87) peptide also binds to the anti-apoptotic protein Bcl-2, albeit with lower binding affinity compared to Bcl-x_L. Here, we demonstrate that a slightly longer Bak peptide corresponding to amino acids 72-89 of Bak binds Bcl-x_L and Bcl-2 equally well. Approximate binding affinity calculations on these peptide-protein complexes confirm the experimental observations. The flow cytometric assay was also used to screen a saturation mutagenesis library of Bak(72-87) variants for improved affinity to Bcl-x_L. The best variants obtained from this library exhibit an apparent K_d to Bcl-x_L 4-fold lower than that of wild-type Bak(72-87).     

Short Term Interactions with Long Term Consequences: Modulation of Chimeric Vessels by Neural Progenitors

Vessels are a critical and necessary component of most tissues, and there has been substantial research investigating vessel formation and stabilization. Several groups have investigated coculturing endothelial cells with a second cell type to promote formation and stabilization of vessels. Some have noted that long-term vessels derived from implanted cocultures are often chimeric consisting of both host and donor cells. The questions arise as to whether the coculture cell might impact the chimeric nature of the microvessels and can modulate the density of donor cells over time. If long-term engineered microvessels are primarily of host origin, any impairment of the host''s angiogenic ability has significant implications for the long-term success of the implant. If one can modulate the host versus donor response, one may be able to overcome a host''s angiogenic impairment. Furthermore, if one can modulate the donor contribution, one may be able to engineer microvascular networks to deliver molecules a patient lacks systemically for long times. To investigate the impact of the cocultured cell on the host versus donor contributions of endothelial cells in engineered microvascular networks, we varied the ratio of the neural progenitors to endothelial cells in subcutaneously implanted poly(ethylene glycol)/poly-L-lysine hydrogels. We found that the coculture of neural progenitors with endothelial cells led to the formation of chimeric host-donor vessels, and the ratio of neural progenitors has a significant impact on the long term residence of donor endothelial cells in engineered microvascular networks in vivo even though the neural progenitors are only present transiently in the system. We attribute this to the short term paracrine signaling between the two cell types. This suggests that one can modulate the host versus donor contributions using short-term paracrine signaling which has broad implications for the application of engineered microvascular networks and cellular therapy more broadly.