Anatomy of protein pockets and cavities: measurement of binding site geometry and implications for ligand design.

Identification and size characterization of surface pockets and occluded cavities are initial steps in protein structure-based ligand design. A new program, CAST, for automatically locating and measuring protein pockets and cavities, is based on precise computational geometry methods, including alpha shape and discrete flow theory. CAST identifies and measures pockets and pocket mouth openings, as well as cavities. The program specifies the atoms lining pockets, pocket openings, and buried cavities; the volume and area of pockets and cavities; and the area and circumference of mouth openings. CAST analysis of over 100 proteins has been carried out; proteins examined include a set of 51 monomeric enzyme-ligand structures, several elastase-inhibitor complexes, the FK506 binding protein, 30 HIV-1 protease-inhibitor complexes, and a number of small and large protein inhibitors. Medium-sized globular proteins typically have 10-20 pockets/cavities. Most often, binding sites are pockets with 1-2 mouth openings; much less frequently they are cavities. Ligand binding pockets vary widely in size, most within the range 10(2)-10(3)A3. Statistical analysis reveals that the number of pockets and cavities is correlated with protein size, but there is no correlation between the size of the protein and the size of binding sites. Most frequently, the largest pocket/cavity is the active site, but there are a number of instructive exceptions. Ligand volume and binding site volume are somewhat correlated when binding site volume is < or =700 A3, but the ligand seldom occupies the entire site. Auxiliary pockets near the active site have been suggested as additional binding surface for designed ligands (Mattos C et al., 1994, Nat Struct Biol 1:55-58). Analysis of elastase-inhibitor complexes suggests that CAST can identify ancillary pockets suitable for recruitment in ligand design strategies. Analysis of the FK506 binding protein, and of compounds developed in SAR by NMR (Shuker SB et al., 1996, Science 274:1531-1534), indicates that CAST pocket computation may provide a priori identification of target proteins for linked-fragment design. CAST analysis of 30 HIV-1 protease-inhibitor complexes shows that the flexible active site pocket can vary over a range of 853-1,566 A3, and that there are two pockets near or adjoining the active site that may be recruited for ligand design.     

Phage display and crystallographic analysis reveals potential substrate/binding site interactions in the protein secretion chaperone CsaA from Agrobacterium tumefaciens

The protein CsaA has been proposed to function as a protein secretion chaperone in bacteria that lack the Sec-dependent protein-targeting chaperone SecB. CsaA is a homodimer with two putative substrate-binding pockets, one in each monomer. To test the hypothesis that these cavities are indeed substrate-binding sites able to interact with other polypeptide chains, we selected a peptide that bound to CsaA from a random peptide library displayed on phage. Presented here is the structure of CsaA from Agrobacterium tumefaciens (AtCsaA) solved in the presence and absence of the selected peptide. To promote co-crystallization, the sequence for this peptide was genetically fused to the amino-terminus of AtCsaA. The resulting 1.65 Å resolution crystal structure reveals that the tethered peptide from one AtCsaA molecule binds to the proposed substrate-binding pocket of a symmetry-related molecule possibly mimicking the interaction between a pre-protein substrate and CsaA. The structure shows that the peptide lies in an extended conformation with alanine, proline and glutamine side chains pointing into the binding pocket. The peptide interacts with the atoms of the AtCsaA-binding pocket via seven direct hydrogen bonds. The side chain of a conserved pocket residue, Arg76, has an “up” conformation when the CsaA-binding site is empty and a “down” conformation when the CsaA-binding site is occupied, suggesting that this residue may function to stabilize the peptide in the binding cavity. The presented aggregation assays, phage-display analysis and structural analysis are consistent with AtCsaA being a general chaperone. The properties of the proposed CsaA-binding pocket/peptide interactions are compared to those from other structurally characterized molecular chaperones.     

Detection of multiscale pockets on protein surfaces using mathematical morphology

Detection of pockets on protein surfaces is an important step toward finding the binding sites of small molecules. In a previous study, we defined a pocket as a space into which a small spherical probe can enter, but a large probe cannot. The radius of the large probes corresponds to the shallowness of pockets. We showed that each type of binding molecule has a characteristic shallowness distribution. In this study, we introduced fundamental changes to our previous algorithm by using a 3D grid representation of proteins and probes, and the theory of mathematical morphology. We invented an efficient algorithm for calculating deep and shallow pockets (multiscale pockets) simultaneously, using several different sizes of spherical probes (multiscale probes). We implemented our algorithm as a new program, ghecom (grid‐based HECOMi finder). The statistics of calculated pockets for the structural dataset showed that our program had a higher performance of detecting binding pockets, than four other popular pocket‐finding programs proposed previously. The ghecom also calculates the shallowness of binding ligands, R_inaccess (minimum radius of inaccessible spherical probes) that can be obtained from the multiscale molecular volume. We showed that each part of the binding molecule had a bias toward a specific range of shallowness. These findings will be useful for predicting the types of molecules that will be most likely to bind putative binding pockets, as well as the configurations of binding molecules. The program ghecom is available through the Web server ( http://biunit.naist.jp/ghecom ). Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Refined 2 A X-ray crystal structure of porcine pancreatic kallikrein A, a specific trypsin-like serine proteinase. Crystallization, structure determination, crystallographic refinement, structure and its comparison with bovine trypsin

Porcine pancreas kallikrein A has been crystallized in the presence of the small inhibitor benzamidine, yielding tetragonal crystals of space group P4₁2₁2 containing two molecules per asymmetric unit. X-ray data up to 2·05 Å resolution have been collected using normal rotation anode as well as synchrotron radiation. The crystal structure of benzamidine-kallikrein has been determined using multiple isomorphous replacement techniques, and has subsequently been refined to a crystallographic R-value of 0·220 by applying a diagonal matrix least-squares energy constraint refinement procedure.Both crystallographically independent kallikrein molecules 1 and 2 are related by a non-integral screw axis and form open, heterologous “dimer” structures. The root-mean-square deviation of both molecules is 0·37 Å for all main-chain atoms. This value is above the estimated mean positional error of about 0·2 Å and reflects some significant conformational differences, especially at surface loops. The binding site of molecule 1 in the asymmetric unit is in contact with residues of molecule 2, whereas the binding site of the latter is free and accessible to the solvent. In both molecules the characteristic “kallikrein loop”, where the peptide chain of kallikrein A is cleaved, is only partially traceable. The carbohydrate attached to Asn95 in this loop, although detectable chemically, is not defined.A comparison of the refined structures of porcine kallikrein and bovine trypsin indicates spatial homology for these enzymes. The root-mean-square difference is 0·68 Å if we compare only main-chain atoms of internal segments. Remarkably large deviations are found in some external loops most of which surround the binding site and form a more compact rampart around it in kallikrein than in trypsin. This feature might explain the strongly reduced activity and accessibility of kallikrein towards large protein substrates and inhibitors (e.g. as shown by the model-building experiments on inhibitor complexes reported by Chen &; Bode. 1983).The conformation of the active site residues is very similar in both enzymes. Tyr99 of kallikrein, which is a leucyl residue in trypsin, protrudes into the binding site and interferes with the binding of peptide substrates (Chen &; Bode. 1983). The kallikrein specificity pocket is significantly enlarged compared with trypsin due to a longer peptide segment, 217 to 220, and to the unique outwards orientation of the carbonyl group of cis-Pro219. Further, the side-chain of Ser226 in porcine kallikrein, which is a glycyl residue in trypsin, partially covers Asp 189 at the bottom of the pocket. These features considerably affect the binding geometry and strength of binding of benzamidine.     

Micelle formation in the presence of photosystem I

The correlation between membrane protein solubilisation and detergent aggregation in aqueous solution is studied for a series of n-alkyl-β-d-maltosides (C_xG₂ with x = 10, 11, 12 being the number of carbon atoms in the alkyl chain) using the trimeric photosystem I core complex (PSIcc) of oxygenic photosynthesis from Thermosynechococcus elongatus as model protein. While protein solubilisation is monitored via the turbidity of the solution, the aggregation behavior of the detergent is probed via the fluorescence spectrum of the polycyclic aromatic hydrocarbon pyrene. In addition, changes of the fluorescence spectrum of PSIcc in response to formation of the detergent belt surrounding its hydrophobic surface are investigated. Solubilisation of PSIcc and aggregation of detergent into micelles or belts are found to be strictly correlated. Both processes are complete at the critical solubilisation concentration (CSC) of the detergent, at which the belts are formed. The CSC depends on the concentration of the membrane protein, [prot], and is related to the critical micelle concentration (CMC) by the empirical law ln(CSC/CMC) = n¯₀ [prot], where the constant n¯₀ = (2.0 ± 0.3) μM⁻¹ is independent of the alkyl chain length x. Formation of protein-free micelles below the CSC is not observed even for x = 10, where a significant excess of detergent is present at the CSC. This finding indicates an influence of PSIcc on micelle formation that is independent of the binding of detergent to the hydrophobic protein surface. The role of the CSC in the optimisation of membrane protein crystallisation is discussed.     

Analysis and prediction of calcium-binding pockets from apo-protein structures exhibiting calcium-induced localized conformational changes

Calcium binding in proteins exhibits a wide range of polygonal geometries that relate directly to an equally diverse set of biological functions. The binding process stabilizes protein structures and typically results in local conformational change and/or global restructuring of the backbone. Previously, we established the MUG program, which utilized multiple geometries in the Ca²⁺‐binding pockets of holoproteins to identify such pockets, ignoring possible Ca²⁺‐induced conformational change. In this article, we first report our progress in the analysis of Ca²⁺‐induced conformational changes followed by improved prediction of Ca²⁺‐binding sites in the large group of Ca²⁺‐binding proteins that exhibit only localized conformational changes. The MUG^SR algorithm was devised to incorporate side chain torsional rotation as a predictor. The output from MUG^SR presents groups of residues where each group, typically containing two to five residues, is a potential binding pocket. MUG^SR was applied to both X‐ray apo structures and NMR holo structures, which did not use calcium distance constraints in structure calculations. Predicted pockets were validated by comparison with homologous holo structures. Defining a “correct hit” as a group of residues containing at least two true ligand residues, the sensitivity was at least 90%; whereas for a “correct hit” defined as a group of residues containing at least three true ligand residues, the sensitivity was at least 78%. These data suggest that Ca²⁺‐binding pockets are at least partially prepositioned to chelate the ion in the apo form of the protein.     

Screening and docking chemical ligands onto pocket cavities of a protease for designing a biocatalyst

A detailed study of the trypsin surface has been carried out to gain insight into its biological functions and interactions which helped to determine the binding specificity. Twenty-four cavity pockets were automatically identified on trypsin from PDB file entry 1AUJ using CASTp (Computed Atlas of Surface Topography of proteins). Molecular docking was exploited as an efficient in silico screening tool for studying protein–ligand interactions. A systematic docking study using Autodock 3.05 has been performed on the five largest binding pockets in trypsin. A set of ten putative chemical ligands was used to dock into selected binding pockets. Docking of ligands into the five largest pockets in trypsin showed that 1,10-phenanthroline and ethanolamine preferentially bound at pocket 24 and benzamidine at pocket 22. Thermodynamically, we also found that ethanol, propanol, propandiol and phosphoethanolamine preferentially bound at pocket 21 whereas p-aminobenzamidine, phenylacetic acid and phenylalanine interacted mainly at pocket 20 based on their lowest interaction free energy.     

Estimation of real-time N load in surface water using dynamic data-driven application system

Agricultural, industrial, and urban activities are the major sources for eutrophication of surface water ecosystems. Currently, determination of nutrients in surface water is primarily accomplished by manually collecting samples for laboratory analysis, which requires at least 24 h. In other words, little to no effort has been devoted to monitoring real-time variations of nutrients in surface water ecosystems due to the lack of suitable and/or cost-effective wireless sensors. However, when considering human health or instantaneous outbreaks such as algal blooms, timely water-quality information is very critical. In this study, we developed a new paradigm of a dynamic data-driven application system (DDDAS) for estimating the real-time loads of nitrogen (N) in a surface water ecosystem. This DDDAS consisted of the following components: (1) a Visual Basic (VB) program for downloading US Geological Survey real-time chlorophyll and discharge data from the internet; (2) a STELLA model for evaluating real-time N loads based on the relationship between chlorophyll and N as well as on river discharge; (3) a batch file for linking the VB program and STELLA model; and (4) a Microsoft Windows Scheduled Task wizard for executing the model and displaying outputs on a computer screen at selected schedules. The DDDAS was validated using field measurements with a very good agreement prior to its applications. Results show that the real-time loads of TN (total N) and NO_x (nitrate and nitrite) varied from positive to negative with the maximums of 1727 kg/h TN and 118 kg/h NO_x and the minimums of −2483 kg/h TN and −168 kg/h NO_x at the selected site. The negative loads occurred because of the back flow of the river in the estuarine environment. Our study suggests that the DDDAS developed in this study was feasible for estimating the real-time variations of TN and NO_x in the surface water ecosystem.     

Crystal Structure of the Pig Pancreatic α-Amylase Complexed with Malto-Oligosaccharides

The structural X-ray map of a pig pancreatic α-amylase crystal soaked (and flash-frozen) with a maltopentaose substrate showed a pattern of electron density corresponding to the binding of oligosaccharides at the active site and at three surface binding sites. The electron density region observed at the active site, filling subsites ?3 through ?1, was interpreted in terms of the process of enzyme-catalyzed hydrolysis undergone by maltopentaose. Because the expected conformational changes in the “flexible loop” that constitutes the surface edge of the active site were not observed, the movement of the loop may depend on aglycone site being filled. The crystal structure was refined at 2.01 å resolution to an R factor of 17.0% (R _free factor of 19.8%). The final model consists of 3910 protein atoms, one calcium ion, two chloride ions, 103 oligosaccharide atoms, 761 atoms of water molecules, and 23 ethylene glycol atoms.     

CX,an algorithm that identifies protruding atoms in proteins

MOTIVATION: A simple and fast algorithm is described that calculates a measure of protrusion (cx) for atoms in protein structures, directly useable with the common molecular graphics programs. RESULTS: A sphere of predetermined radius is centered around each non-hydrogen atom, and the volume occupied by the protein and the free volume within the sphere (internal and external volumes, respectively) are calculated. Atoms in protruding regions have a high ratio (cx) between the external and the internal volume. The program reads a PDB file, and writes the output in the same format, with cx values in the B factor field. Output structure files can be directly displayed with standard molecular graphics programs like RASMOL, MOLMOL, Swiss-PDB Viewer and colored according to cx values. We show the potential use of this program in the analysis of two protein-protein complexes and in the prediction of limited proteolysis sites in native proteins. AVAILABILITY: The algorithm is implemented in a standalone program written in C and its source is freely available at ftp.icgeb.trieste.it/pub/CX or on request from the authors.     

Structures of Trypanosoma brucei Methionyl-tRNA Synthetase with Urea-Based Inhibitors Provide Guidance for Drug Design against Sleeping Sickness

Methionyl-tRNA synthetase of Trypanosoma brucei (TbMetRS) is an important target in the development of new antitrypanosomal drugs. The enzyme is essential, highly flexible and displaying a large degree of changes in protein domains and binding pockets in the presence of substrate, product and inhibitors. Targeting this protein will benefit from a profound understanding of how its structure adapts to ligand binding. A series of urea-based inhibitors (UBIs) has been developed with IC₅₀ values as low as 19 nM against the enzyme. The UBIs were shown to be orally available and permeable through the blood-brain barrier, and are therefore candidates for development of drugs for the treatment of late stage human African trypanosomiasis. Here, we expand the structural diversity of inhibitors from the previously reported collection and tested for their inhibitory effect on TbMetRS and on the growth of T. brucei cells. The binding modes and binding pockets of 14 UBIs are revealed by determination of their crystal structures in complex with TbMetRS at resolutions between 2.2 Å to 2.9 Å. The structures show binding of the UBIs through conformational selection, including occupancy of the enlarged methionine pocket and the auxiliary pocket. General principles underlying the affinity of UBIs for TbMetRS are derived from these structures, in particular the optimum way to fill the two binding pockets. The conserved auxiliary pocket might play a role in binding tRNA. In addition, a crystal structure of a ternary TbMetRS•inhibitor•AMPPCP complex indicates that the UBIs are not competing with ATP for binding, instead are interacting with ATP through hydrogen bond. This suggests a possibility that a general ‘ATP-engaging’ binding mode can be utilized for the design and development of inhibitors targeting tRNA synthetases of other disease-causing pathogen.     

5f-Element complexes with a p-tert-butylcalix[4]arene bearing phosphinoyl pendant arms: Separation from rare earths and structural studies

Phosphinoylated calixarenes feature high coordination ability toward f elements and a great potentiality toward actinide/rare earth separation. Here, we report three characteristic properties of a tetra-phosphinoylated p-tert-butylcalix[4]arene, B₄bL⁴ functionalized with phosphinoyl pendant arms: (i) its coordination ability toward Th(IV) complexation in organic medium, (ii) its ability to separate thorium from yttrium, lanthanum, and europium in three different organic media, and (iii) the X-ray crystal structure of the La complex. Thorium(IV) forms 1:1 and 1:2 (M:L) complexes with B₄bL⁴: Th(NO₃)₄(B₄bL⁴)_n·xH₂O (n = 1, x = 1, 1; n = 2, x = 4, 2). Spectroscopic data point to the inner coordination sphere of 1 and 2 containing nitrate ions and water molecules. Molecular modeling of 1 yielded an 8-coordinate species and its coordination polyhedron can be described as a distorted square antiprism while that for 2, a 9-coordinate species, as a distorted tricapped trigonal prism. The extraction study of tetravalent thorium and trivalent rare-earth (Y, La, Eu) ions from acidic nitrate media by B₄bL⁴ in chloroform shows thorium being much more extracted than the rare earths, with selectivity close to 100%. The extraction behavior can be easily modulated by changing the initial conditions (pH, nitrate concentration). The X-ray structure of [LaB₄bL⁴(H₂O)₅] CH₃CN·(ClO₄)₃ points to the La^III ion lying on a C₄ axis and being 9-coordinated by the four O(P) atoms and five O atoms from water molecules. It is located in the middle of the void formed by the four O-CH₂-PO(Me)₂ pendant arms.     

Phloroglucinol-bridged trinuclear complexes with three paramagnetic octahedral nickel(II) ions: Syntheses, crystal structures, and magnetic properties

Three new C₃-symmetric tritopic ligands with a central phloroglucinol bridging unit have been synthesized and characterized. The ligands are accessible through Schiff-base condensation of 2,4,6-triformylphloroglucinol with 2-aminomethylpyridine (H₃tfpg-ampy), N,N-bis(pyridin-2-ylmethyl)-ethylenediamine (H₃tfpg-unspenp), and benzhydrazide (H₆tfpg-bhy). These ligands differ in nature and number of the donor atoms within the resulting binding pockets. Based on these ligands the synthesis of the first trinuclear phloroglucinol-bridged nickel(II) complexes with three octahedrally coordinated nickel centers is reported. The ligands H₃tfpg-ampy and H₆tfpg-bhy, which provide tridentate binding pockets, react with nickel(II) perchlorate in the presence of bis(pyridin-2-ylethyl)-amine (bpea) as an additional tridentate capping ligand leading to the formation of the trinuclear complexes [Ni₃(tfpg-ampy)(bpea)₃](ClO₄)₃ and [Ni₃(tfpg-bhy)(bpea)₃](ClO₄)₃, respectively. Due to the pentadentate binding pocket in ligand H₃tfpg-unspenp, no additional coligand is needed and a water molecule occupies the sixth coordination site at the nickel(II) ion resulting in the complex [Ni₃(tfpg-unspenp)(H₂O)₃](ClO₄)₃. Temperature-dependent magnetic measurements reveal overall weak antiferromagnetic exchange interactions within the trinuclear complex together with a rather strong zero-field splitting (ZFS) for the nickel(II) ions. The observed isotropic coupling constants for the three complexes are in the range of 0.14 < − J < 0.37 cm⁻¹, whereas for the zero-field splitting parameter ∣D∣ values between 1.8 and 5.5 cm⁻¹ are found. This is indicative for competitive spin-polarization and superexchange mechanisms, with the latter prevailing the interaction between the nickel(II) ions through the meta-phenylene-linkage for the complexes reported.     

Preparation of carbonitride films in the active and afterglow phases of a glow discharge

The formation of carbonitride (C _x N _y ) films in the active and afterglow phases of a glow discharge in CH₄-N₂ mixtures (as well in these mixtures diluted with argon and helium) was studied experimentally. The dependences of the film growth rate on the discharge current and gas pressure are obtained. The composition (the N/C ratio) and IR absorption spectra of the films are determined. Measurements of the absorption spectra made it possible to identify bonds between C and N atoms. A novel method of carbonitride film deposition in the “double afterglow” mode was proposed. The use of this method appreciably increases the film deposition rate. Possible mechanisms of the formation and destruction of carbonitride films in the active and afterglow phases of the discharge are discussed.     

Estimating Orientation Factors in the FRET Theory of Fluorescent Proteins: The TagRFP-KFP Pair and Beyond

The orientation factor κ², one of the key parameters defining Förster resonance energy transfer efficiency, is determined by the transition dipole moment orientations of the donor and acceptor species. Using the results of quantum chemical and quantum mechanical/molecular mechanical calculations for the chromophore-containing pockets in selected colored proteins of the green fluorescent protein family, we derived transition dipole moments corresponding to the S_0,min → S₁ excitation for green fluorescent protein, red fluorescent protein (TagRFP), and kindling fluorescent protein, and the S_1,min → S₀ emission for TagRFP. These data allowed us to estimate κ² values for the TagRFP-linker-kindling fluorescent protein tetrameric complex required for constructing novel sensors.     

Magnesium (II) poly(pyrazolyl)borate derivatives - Synthesis, spectral and structural studies

A series of magnesium complexes of general formula [Mg(Tp^x)₂] (Tp^x = Tp, Tp^∗, Tp^∗Cl, pzTp) and [Mg(Tp^x)X] (X = Cl, Tp^x = Tp^tBu or pz⁰Tp^p-Tol; X = acetate, Tp^x = Tp^tBu) were synthesised from magnesium chloride or acetate and M(Tp^x) (M = K, Na or Tl) in dichloromethane or alcoholic solution. These compounds are air-stable solids, sparingly soluble in most organic solvents; they have been characterized by elemental analysis, IR, ¹H and ¹³C NMR spectra and, in selected cases, also by conductivity and molecular weight measurements. Single crystal X-ray diffraction studies of [Mg(Tp)₂], [Mg(Tp*)₂] and [Mg(Tp*^Cl)₂] show unsolvated neutral bis(tripod ligand)magnesium(II) molecules with six-coordinate magnesium atoms (〈Mg-N〉 2.167(6), 2.19(2), 2.205(4) Å).     

Lactoferrin,myeloperoxidase, and ceruloplasmin: complementary gearwheels cranking physiological and pathological processes

Copper-containing plasma protein ceruloplasmin (Cp) forms a complex with lactoferrin (Lf), an iron-binding protein, and with the heme-containing myeloperoxidase (Mpo). In case of inflammation, Lf and Mpo are secreted from neutrophil granules. Among the plasma proteins, Cp seems to be the preferential partner of Lf and Mpo. After an intraperitoneal injection of Lf to rodents, the “Cp–Lf” complex has been shown to appear in their bloodstream. Cp prevents the interaction of Lf with protoplasts of Micrococcus luteus. Upon immunoprecipitation of Cp, the blood plasma becomes depleted of Lf and in a dose-dependent manner loses the capacity to inhibit the peroxidase activity of Mpo, but not the Mpo-catalyzed oxidation of thiocyanate in the (pseudo)halogenating cycle. Antimicrobial effect against E. coli displayed by a synergistic system that includes Lf and Mpo–H₂O₂–chloride, but not thiocyanate, as the substrate for Mpo is abrogated when Cp is added. Hence, Cp can be regarded as an anti-inflammatory factor that restrains the halogenating cycle and redirects the synergistic system Mpo–H₂O₂–chloride/thiocyanate to production of hypothiocyanate, which is relatively harmless for the human organism. Structure and functions of the “2Cp–2Lf–Mpo” complex and binary complexes Cp–Lf and 2Cp–Mpo in inflammation are discussed.