High-resolution neutron and X-ray diffraction room-temperature studies of an H-FABP-oleic acid complex: study of the internal water cluster and ligand binding by a transferred multipolar electron-density distribution

Crystal diffraction data of heart fatty acid binding protein (H-FABP) in complex with oleic acid were measured at room temperature with high-resolution X-ray and neutron protein crystallography (0.98 and 1.90 Å resolution, respectively). These data provided very detailed information about the cluster of water molecules and the bound oleic acid in the H-FABP large internal cavity. The jointly refined X-ray/neutron structure of H-FABP was complemented by a transferred multipolar electron-density distribution using the parameters of the ELMAMII library. The resulting electron density allowed a precise determination of the electrostatic potential in the fatty acid (FA) binding pocket. Bader's quantum theory of atoms in molecules was then used to study interactions involving the internal water molecules, the FA and the protein. This approach showed H···H contacts of the FA with highly conserved hydrophobic residues known to play a role in the stabilization of long-chain FAs in the binding cavity. The determination of water hydrogen (deuterium) positions allowed the analysis of the orientation and electrostatic properties of the water molecules in the very ordered cluster. As a result, a significant alignment of the permanent dipoles of the water molecules with the protein electrostatic field was observed. This can be related to the dielectric properties of hydration layers around proteins, where the shielding of electrostatic interactions depends directly on the rotational degrees of freedom of the water molecules in the interface.Facultad de Ciencias ExactasInstituto de Física de Líquidos y Sistemas Biológico

Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants

Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity

High-resolution neutron and X-ray diffraction room-temperature studies of an H-FABP-oleic acid complex: study of the internal water cluster and ligand binding by a transferred multipolar electron-density distribution

Crystal diffraction data of heart fatty acid binding protein (H-FABP) in complex with oleic acid were measured at room temperature with high-resolution X-ray and neutron protein crystallography (0.98 and 1.90 Å resolution, respectively). These data provided very detailed information about the cluster of water molecules and the bound oleic acid in the H-FABP large internal cavity. The jointly refined X-ray/neutron structure of H-FABP was complemented by a transferred multipolar electron-density distribution using the parameters of the ELMAMII library. The resulting electron density allowed a precise determination of the electrostatic potential in the fatty acid (FA) binding pocket. Bader's quantum theory of atoms in molecules was then used to study interactions involving the internal water molecules, the FA and the protein. This approach showed H···H contacts of the FA with highly conserved hydrophobic residues known to play a role in the stabilization of long-chain FAs in the binding cavity. The determination of water hydrogen (deuterium) positions allowed the analysis of the orientation and electrostatic properties of the water molecules in the very ordered cluster. As a result, a significant alignment of the permanent dipoles of the water molecules with the protein electrostatic field was observed. This can be related to the dielectric properties of hydration layers around proteins, where the shielding of electrostatic interactions depends directly on the rotational degrees of freedom of the water molecules in the interface.Facultad de Ciencias ExactasInstituto de Física de Líquidos y Sistemas Biológico

Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants

Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity.PMC529941

Structure of the E6/E6AP/p53 complex required for HPV-mediated degradation of p53

The p53 pro-apoptotic tumor suppressor is mutated or functionally altered in most cancers. In epithelial tumors induced by “high-risk” mucosal Human Papillomaviruses (hrm-HPVs), including human cervical carcinoma and a growing number of head-and-neck cancers (1), p53 is degraded by the viral oncoprotein E6 (2). In this process, E6 binds to a short LxxLL consensus sequence within the cellular ubiquitin ligase E6AP (3). Subsequently, the E6/E6AP heterodimer recruits and degrades p53 (4). Neither E6 nor E6AP are separately able to recruit p53 (3,5), and the precise mode of assembly of E6, E6AP and p53 is unknown. Here, we solved the crystal structure of a ternary complex comprising full-length HPV16 E6, the LxxLL motif of E6AP and the core domain of p53. The LxxLL motif of E6AP renders the conformation of E6 competent for interaction with p53 by structuring a p53-binding cleft on E6. Mutagenesis of critical positions at the E6-p53 interface disrupts p53 degradation. The E6-binding site of p53 is distal from previously described DNA- and protein-binding surfaces of the core domain. This suggests that, in principle, E6 may avoid competition with cellular factors by targeting both free and bound p53 molecules. The E6/E6AP/p53 complex represents a prototype of viral hijacking of both the ubiquitin-mediated protein degradation pathway and the p53 tumor suppressor pathway. The present structure provides a framework for the design of inhibitory therapeutic strategies against HPV-mediated oncogenesis

Identification of a novel polyfluorinated compound as a lead to inhibit human enzymes aldose reductase and AKR1B10 : structure determination of both ternary complexes and implications for drug design

Aldo-keto reductases (AKRs) are mostly monomeric enzymes which fold into a highly conserved ([alpha]/[beta])8 barrel, while their substrate specificity and inhibitor selectivity are determined by interaction with residues located in three highly variable external loops. The closely related human enzymes aldose reductase (AR or AKR1B1) and AKR1B10 are of biomedical interest because of their involvement in secondary diabetic complications (AR) and in cancer, e.g. hepatocellular carcinoma and smoking-related lung cancer (AKR1B10). After characterization of the IC50 values of both AKRs with a series of polyhalogenated compounds, 2,2',3,3',5,5',6,6'-octafluoro-4,4'-biphenyldiol (JF0064) was identified as a lead inhibitor of both enzymes with a new scaffold (a 1,1'-biphenyl-4,4'-diol). An ultrahigh-resolution X-ray structure of the AR-­NADP+-JF0064 complex has been determined at 0.85 Å resolution, allowing it to be observed that JF0064 interacts with the catalytic residue Tyr48 through a negatively charged hydroxyl group (i.e. the acidic phenol). The non-competitive inhibition pattern observed for JF0064 with both enzymes suggests that this acidic hydroxyl group is also present in the case of AKR1B10. Moreover, the combination of surface lysine methylation and the introduction of K125R and V301L mutations enabled the determination of the X-ray crystallo­graphic structure of the corresponding AKR1B10-NADP+-JF0064 complex. Comparison of the two structures has unveiled some important hints for subsequent structure-based drug-design efforts

Full-length human placental sFlt-1-e15a isoform induces distinct maternal phenotypes of preeclampsia in mice

<div><p>Objective</p><p>Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring.</p><p>Methods</p><p>Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia.</p><p>Results</p><p>Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3±51.7μg/mg vs. 19.3±5.6μg/mg, p = 4.4x10^-2; GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2x10^-2). Placental and fetal weights did not differ between the groups. One mouse with liver disease developed early-onset preeclampsia-like symptoms with intrauterine growth restriction (IUGR).</p><p>Conclusions</p><p>A mouse model of late-onset preeclampsia was developed with the overexpression of hsFlt-1-e15a, verifying the <i>in vivo</i> pathologic effects of this primate-specific, predominant placental sFlt-1 isoform. HsFlt-1-e15a induced early-onset preeclampsia-like symptoms associated with IUGR in a mouse with a liver disease. Our findings support that hsFlt-1-e15a is central to the terminal pathway of preeclampsia, and it can induce the full spectrum of symptoms in this obstetrical syndrome.</p></div

Improving Crystallographic Macromolecular Images: The Real-Space Approach

Macromolecular crystallographicy is a unique tool for imaging the structures of proteins and nucleic acids. Images are obtained from the Fourier transform of the diffraction pattern of the crystal by use of X-ray, neutron, and / or electron scattering. When X-ray and neutron scattering are used only diffraction amplitudes are experimentally measured, and phases have to be obtained. Multiple isomorphous replacement (MIR) has been the technique of choice for solving this phase problem in the determination of most macromolecular structures. Unfortunately, the method is extremely time consuming, especially when compared with the solution techniques available for small molecules; moreover, structure solution by MIR, even after many years of work, is hardly guaranteed. These drawbacks have stimulated efforts to enhance MIR as a phasing technique. The methods discussed in this paper (without exhaustive coverage, owing to space limitations) have so far been used to refine and/or to extend MIR phases, and also to open up the possibility of ab initio phase determination. Following the early fundamental work of Karle & Hauptman (34, 35) and Sayre (60), reciprocal-space direct methods were applied to solve the structure of the majority of small molecules (via widely used packages, e.g. MULTAN and SHELX). These methods are used to derive phases statistically from the atomic character of the density. The extension of these methods to macromolecular crystallography is beyond the scope of this review. Macromolecules present a more difficult problem. The diffraction data are rarely obtained at high enough resolution for the application of the atomicity constraint. Also, the accuracy of the phase predictions by reciprocal-space direct methods decreases with the size of the molecule. However, there are other a priori physical constraints applicable to macro-molecular density functions, e.g. continuity and solvent flatness. These constraints are more readily expressed in real space than in reciprocal space. Procedures that exploit such physical constraints in real space are commonly known as density modification (DM) methods. These techniques do not merely consist of real space imposition of a priori physical constraints, but also include reciprocal-space steps of comparable importance. These mixed real and reciprocal-space DM algorithms are the main subject of this review

Ab initio low-resolution phasing in crystallography of macromolecules by maximization of likelihood

International audienceStatistical likelihood criteria were tested to select the true (or closest to true) structure-factor phases from an ensemble of phase sets. To define the criterion value for a given trial phase set, the trial `molecular region' is defined as a region consisting of the points with the highest values in the Fourier synthesis calculated with the observed magnitudes and the trial set of phases. The structure studied is considered as composed of atoms randomly placed inside the trial molecular region. The figure of merit is defined as the likelihood corresponding to this hypothesis, i.e. the probability that the structure-factor magnitudes calculated (from the positions of atoms randomly placed into the trial region) are equal to the observed magnitudes. The concept of generalized likelihood is introduced to make the calculations more straightforward. The tests performed for known structures with the use of experimentally observed magnitudes show that in general it is impossible to unambiguously determine the best phases among a `population' of trial phase sets. Nevertheless, the random generation of a great number of phase sets and the selection of phase sets with high likelihood values give a collection of variants with a higher concentration of `good' phase sets than those found in the original population. Averaging the selected phase sets gives a starting solution of the low-resolution phase problem

Neutron protein crystallography: hydrogen protons and hydration in bio-macromolecules

This text is dedicated to the emerging field of neutron protein crystallography (NPC). It covers all of the practical aspects of NPC and demonstrates how NPC can explore protein features such as hydrogen bonds, protonation and deprotonation of amino acid residues, and hydration structures