Structure of ethanol-inhibited porcine pepsin at 2-A resolution and binding of the methyl ester of phenylalanyl-diiodotyrosine to the enzyme

An account of x-ray crystallographic studies of monoclinic porcine pepsin crystals is presented. The chain fold specific for aspartyl proteases is described in detail. As the results of 2-A refinement have shown, the actual structure is that of ethanol-inhibited pepsin. The structure, although close to those of fungal aspartyl proteases, has some specific features: one of them is an insertion near the S'1 site which restricts the position of dipeptide substrates and makes their productive binding more probable than in the fungal enzymes. 3-A resolution data on the binding of the dipeptide phenylalanyl-diiodotyrosine methyl ester are discussed.     

Accessibility and order of water sites in and around proteins: A crystallographic time-averaging study.

Water plays an essential role in most biological processes. Water molecules solvating biomolecules are generally in fast exchange with the environment. Nevertheless, well-defined electron density is seen for water associated with proteins whose crystal structure is determined to high resolution. The relative accessibility of these water sites is likely to be relevant to their biological role but is difficult to assess. A time-averaging crystallographic refinement simulation on basic pancreatic trypsin inhibitor successfully characterizes the relative accessibility of the crystallographic water sites. In such a refinement simulation water diffuses through the crystal lattice in a manner that is consistent with the crystallographic data. This refinement discovers that internal crystallographic waters in this particular protein are bridged to the outside protein surface via a series of progressively more accessible water sites. On the surface of the protein, water molecules exchange quickly between crystallographic water sites. Time-averaging crystallographic refinement provides a view based on experimental data of the relative accessibility of water sites in and around a protein in a crystalline environment. Proteins 1999;36:501-511.     

Molecular and crystal structures of monoclinic porcine pepsin refined at 1.8 A resolution

The molecular structure of the archetypal aspartic proteinase, porcine pepsin (EC 3.4.23.1), has been refined using data collected from a single monoclinic crystal on a twin multiwire detector system to 1.8 A resolution. The current crystallographic R-factor (= sigma parallel to Fo/-/Fc parallel to/sigma/Fo/) is 0.174 for the 20,519 reflections with /Fo/ greater than or equal to 3 sigma (Fo) in the range 8.0 to 1.8 A (/Fo/ and /Fc/ are the observed and calculated structure factor amplitudes respectively). The refinement has shown conclusively that there are only 326 amino acid residues in porcine pepsin. Ile230 is not present in the molecule. The two catalytic residues Asp32 and Asp215 have dispositions in porcine pepsin very similar to the dispositions of the equivalent residues in the other aspartic proteinases of known structure. A bound solvent molecule is associated with both carboxyl groups at the active site. No bound ethanol molecule could be identified conclusively in the structure. The average thermal motion parameter of the residues that comprise the C-terminal domain of pepsin is approximately twice that of the residues in the N-terminal domain. Comparisons of the tertiary structure of pepsin with porcine pepsinogen, penicillopepsin, rhizopus pepsin and endothia pepsin reveal that the N-terminal domains are topographically more similar than the conformationally flexible C-terminal domains. The conformational differences may be modeled as rigid-body movements of "reduced" C-terminal domains (residues 193 to 212 and 223 to 298 in pepsin numbering). A similar movement of the C-terminal domain of endothia pepsin has been observed upon inhibitor binding. A phosphoryl group covalently attached to Ser68 O gamma has been identified in the electron density map of porcine pepsin. The low pKa1 value for this group, coupled with unusual microenvironments for several of the aspartyl carboxylate groups, ensures a net negative charge on porcine pepsin in a strongly acid medium. Thus, there is a structural explanation for the very early observations of "anodic migration" of porcine pepsin at pH 1. In the crystals, the molecules are packed tightly into a monoclinic unit cell. There are 190 direct contacts (less than or equal to 4.0 A) between a central pepsin molecule and the five unique symmetry-related molecules surrounding it in the crystalline lattice. The tight packing in this cell makes pepsin's active site and binding cleft relatively inaccessible to substrate analogs or inhibitors.     

Comparison of pepsins isolated from porcine, bovine and Penicillium jathiuellum

1. The reactivities of pepsins, isolated from three different sources (porcine, bovine, and Penicillium jathiuelum), toward ester and peptide substrates were compared. 2. Porcine pepsin showed the highest activity followed by penicillopepsin with bovine pepsin being the least active. 3. The esterase activity of penicillopepsin was greater than that of porcine pepsin with bovine pepsin again showing the least activity. 4. The CD spectra indicate that porcine and bovine pepsin have similar conformations, even though bovine pepsin shows less ellipticity at 220 nm. 5. Penicillopepsin showed a completely opposite sign in the near-u.v. region of the CD spectrum. 6. The far-u.v. region of the CD spectrum of penicillopepsin strongly suggests a beta-sheet structure. 7. Previously reported X-ray crystallographic data suggest that porcine pepsin has a compact three-dimensional structure, while the structures of bovine and penicillopepsins are partially unfolded.     

Normal mode refinement: crystallographic refinement of protein dynamic structure applied to human lysozyme.

A new method of dynamic structure refinement of protein x-ray crystallography, normal mode refinement, is developed. In this method the Debye-Waller factor is expanded in terms of the low-frequency normal modes and external normal modes, whose amplitudes and couplings are optimized in the process of crystallographic refinement. By this method, internal and external contributions to the atomic fluctuations can be separated. Also, anisotropic atomic fluctuations and their interatomic correlations can be determined experimentally even with a relatively small number of adjustable parameters. The method is applied to the analysis of experimental data of human lysozyme to reveal its dynamic structure.     

X-ray study of chymosin. I. Molecular replacement at a 3 angstroms resolution

The determination of the three dimensional structure of chymosin at 3 A resolution by molecular replacement method is described. The rotation functions for various aspartic proteases were calculated and combined results were used for the refinement of orientational parameters of chymosin molecules in the unit cell. The interpretation of Crowther-Blow translation function map with packing consideration enable to place correctly the molecules in the chymosin unit cell. Several difference Fourier syntheses for chymosin were calculated and differences between pepsin and chymosin structures were detected.     

Normal mode refinement: crystallographic refinement of protein dynamic structure. I. Theory and test by simulated diffraction data.

A dynamic structure refinement method for X-ray crystallography, referred to as the normal mode refinement, is proposed. The Debye-Waller factor is expanded in terms of the low-frequency normal modes whose amplitudes and eigenvectors are experimentally optimized in the process of the crystallographic refinement. In this model, the atomic fluctuations are treated as anisotropic and concerted. The normal modes of the external motion (TLS model) are also introduced to cover the factors other than the internal fluctuations, such as the lattice disorder and diffusion. A program for the normal mode refinement (NM-REF) has been developed. The method has first been tested against simulated diffraction data for human lysozyme calculated by a Monte Carlo simulation. Applications of the method have demonstrated that the normal mode refinement has: (1) improved the fitting to the diffraction data, even with fewer adjustable parameters; (2) distinguished internal fluctuations from external ones; (3) determined anisotropic thermal factors; and (4) identified concerted fluctuations in the protein molecule.     

Crystal structure of human pepsin and its complex with pepstatin.

The three-dimensional crystal structure of human pepsin and that of its complex with pepstatin have been solved by X-ray crystallographic methods. The native pepsin structure has been refined with data collected to 2.2 A resolution to an R-factor of 19.7%. The pepsin:pepstatin structure has been refined with data to 2.0 A resolution to an R-factor of 18.5%. The hydrogen bonding interactions and the conformation adopted by pepstatin are very similar to those found in complexes of pepstatin with other aspartic proteinases. The enzyme undergoes a conformational change upon inhibitor binding to enclose the inhibitor more tightly. The analysis of the binding sites indicates that they form an extended tube without distinct binding pockets. By comparing the residues on the binding surface with those of the other human aspartic proteinases, it has been possible to rationalize some of the experimental data concerning the different specificities. At the S1 site, valine at position 120 in renin instead of isoleucine, as in the other enzymes, allows for binding of larger hydrophobic residues. The possibility of multiple conformations for the P2 residue makes the analysis of the S2 site difficult. However, it is possible to see that the specific interactions that renin makes with histidine at P2 would not be possible in the case of the other enzymes. At the S3 site, the smaller volume that is accessible in pepsin compared to the other enzymes is consistent with its preference for smaller residues at the P3 position.     

X-ray analyses of aspartic proteinases. IV. Structure and refinement at 2.2 A resolution of bovine chymosin

The structure of calf chymosin (EC 3.4.23.3), the aspartic proteinase from the gastric mucosa, was solved using the technique of molecular replacement. We describe the use of different search models based on distantly related fungal aspartic proteinases and investigate the effect of using only structurally conserved regions. The structure has been refined to a crystallographic R-factor of 17% at 2.2 A resolution with an estimated co-ordinate error of 0.21 A. In all, 136 water molecules have been located of which eight are internal. The structure of chymosin resembles that of pepsin and other aspartic proteinases. However, there is a considerable rearrangement of the active-site "flap" and, in particular, Tyr75 (pepsin numbering), which forms part of the specificity pockets S1 and S1'. This is probably a consequence of crystal packing. Electrostatic interactions on the edge of the substrate binding cleft appear to account for the restricted proteolysis of the natural substrate kappa-casein by chymosin. The local environment of invariant residues is examined, showing that structural constraints and side-chain hydrogen bonding can play an important role in the conservation of particular amino acids.     

Three-dimensional structure of an Fv from a human IgM immunoglobulin.

An IgM(kappa) immunoglobulin from a patient (Pot) with Waldenstrom's macroglobulinemia was hydrolyzed with pepsin to release a fragment consisting of the 'variable' (V) domains of the light and heavy chains plus eight residue 'tails' from the 'constant' (C) domains. The crystal structure of this fragment was determined at 2.3 A resolution by molecular replacement and crystallographic refinement methods. When examined separately, the light chain component closely resembles another human kappa chain (Rei) in both the beta-pleated sheet regions and the 'hypervariable' loops. The conserved pleated sheets in the heavy chain are similar to those in the human Kol IgG1 protein, but the third hypervariable loop in particular is different from that in any immunoglobulin structure described to date. As in the Kol protein, this loop blocks the access to any internal active site along the light-heavy chain interface. Unlike the Kol protein, however, the loop does not protrude beyond the boundaries of a conventional antigen combining site. Instead, it forms a very compact structure, which fills almost all residual space between the domains. This is an example of one dominant complementarity-determining region (CDR) essentially negating the diversity possible with five other CDRs in the two chains. Ordered water molecules are associated with light chain constituents along the interface, but not with CDR3 of the heavy chain. In screening exercises the Pot IgM failed to bind a wide variety of peptides. Together, the results suggest that ligand binding can only occur on external surfaces of the protein. These surfaces carry a limited number of side chains usually assigned to CDRs in more typical antibodies.     

Effect of anisotropy and anharmonicity on protein crystallographic refinement. An evaluation by molecular dynamics

Molecular dynamics simulations are employed to determine the errors introduced by anharmonicity and anisotropy in the structure and temperature factors obtained for proteins by refinement of X-ray diffraction data. Simulations (25 ps and 300 ps) of metmyoglobin are used to generate time-averaged diffraction data at 1.5 A resolution. The crystallographic restrained-parameter least-squares refinement program PROLSQ is used to refine models against these simulated data. The resulting atomic positions and isotropic temperature factors are compared with the average structure and fluctuations calculated directly from the simulations. It is found that significant errors in the atomic positions and fluctuations are introduced by the refinement, and that the errors increase with the magnitude of the atomic fluctuations. Of particular interest is the fact that the refinement generally underestimates the atomic motions. Moreover, while the actual fluctuations go up to a mean-square value of about 5 A2, the X-ray results never go above approximately 2 A2. This systematic deviation in the motional parameters appears to be due to the use of a single-site isotropic model for the atomic fluctuations. Many atoms have multiple peaks in their probability distribution functions. For some atoms, the multiple peaks are seen in difference electron density maps and it is possible to include these in the refinement as disordered residues. However, for most atoms the refinement fits only one peak and neglects the rest, leading to the observed errors in position and temperature factor. The use of strict stereochemical restraints is inconsistent with the average dynamical structure; nevertheless, refinement with tight restraints results in structures that are comparable to those obtained with loose restraints and better than those obtained with no restraints. The results support the use of tight stereochemical restraints, but indicate that restraints on the variation of temperature factors are too restrictive.     

Further refinement of the structure of yeast tRNAPhe.

We have refined the monoclinic crystal structure of yeast tRNA^Phe against a complete set of X-ray data at 2.5 Å resolution, using real-space refinement and a combination of energy minimisation and crystallographic least-squares. This refinement has allowed us to define the conformation of residue D16, and to make corrections to Y37 and A76. We have found an additional magnesium binding site (making a total of four), a number of water molecules, and a possible spermine molecule.A revised list of torsion angles is given.     

Sequencing a protein by x-ray crystallography. II. Refinement of yeast hexokinase B co-ordinates and sequence at 2.1 A resolution.

Although the amino acid sequence of yeast hexokinase B has not been determined by chemical means, crystallographic refinement of the hexokinase monomer was carried out at 2.1 Å resolution to improve both the atomic co-ordinates and the amino acid sequence, which had been obtained from a 2.5 Å electron density map. The atomic co-ordinates were adjusted by real-space refinement into a multiple isomorphous replacement map, followed by automated difference Fourier refinement, and restrained parameter structure factor least-squares refinement. The amino acid sequence was altered periodically after visual inspection of (F_o ? F_c) difference electron density maps. Evidence of the improvement in the amino acid sequence was provided by the better agreement between the X-ray and chemically derived amino acid compositions, and most importantly by the ability to locate two short peptides which had been chemically sequenced. While only 6 out of the 18 residues in these two peptides agree with the sequence of the original model, 12 residues agree with the sequence of the refined model and the others differ by only an atom or two. The refined model contains 3293 of of the 3596 non-hydrogen atoms expected from the amino acid composition and 152 bound water molecules. The crystallographic R factor at 2.1 Å is 0.25.We show that there are several advantages to refining the structure of even a protein of unknown sequence. (1) Improved phases can be obtained to the resolution limit of the diffraction pattern starting with a model derived from a 2.5 Å map. (2) The accuracy of the amino acid sequence derived by X-ray methods alone can be substantially improved. (3) Functionally important residues can be identified before chemical sequence information is available. (4) The improved X-ray sequence should greatly reduce the effort required to obtain a chemical sequence; since peptides as short as eight or nine residues can be located in the refined X-ray sequence, peptides do not need to be overlapped by chemical means.     

Hydration of the DNA bases is local.

Ordered hydration sites were determined for the nucleotide bases in B-type conformations using the crystal structure data on 14 B-DNA decamer structures. A method of density representation was extended so that positions, occupancies, and distributions of the hydration sites were predicted around a B-DNA double helix by a method analogous to crystallographic refinement. The predicted hydration sites correctly reproduce the main features of hydration around the B-DNA dodecamer. In contrast to the previous observations, the newly available crystal data show the same extent of hydration of guanine and adenine, and of cytosine and thymine.     

Using molecular dynamics for the refinement of atomistic models of GPCRs by homology modeling

Despite GPCRs sharing a common seven helix bundle, analysis of the diverse crystallographic structures available reveal specific features that might be relevant for ligand design. Despite the number of crystallographic structures of GPCRs steadily increasing, there are still challenges that hamper the availability of new structures. In the absence of a crystallographic structure, homology modeling remains one of the important techniques for constructing 3D models of proteins. In the present study we investigated the use of molecular dynamics simulations for the refinement of GPCRs models constructed by homology modeling. Specifically, we investigated the relevance of template selection, ligand inclusion as well as the length of the simulation on the quality of the GPCRs models constructed. For this purpose we chose the crystallographic structure of the rat muscarinic M3 receptor as reference and constructed diverse atomistic models by homology modeling, using different templates. Specifically, templates used in the present work include the human muscarinic M2; the more distant human histamine H1 and the even more distant bovine rhodopsin as shown in the GPCRs phylogenetic tree. We also investigated the use or not of a ligand in the refinement process. Hence, we conducted the refinement process of the M3 model using the M2 muscarinic as template with tiotropium or NMS docked in the orthosteric site and compared with the results obtained with a model refined without any ligand bound.     

Crystal structure of yeast phenylalanine transfer RNA. II. Structural features and functional implications.

The structural features of yeast phenylalanine transfer RNA are analyzed and documented in detail, based on atomic co-ordinates obtained from an extensive crystallographic refinement of the crystal structure of the molecule at 2.7 Å resolution (see preceding paper). We describe here: the relative orientation and the helicity of the base-paired stems; more definitive assignments of tertiary hydrogen bonds involving bases, riboses and phosphates; binding sites for magnesium hydrates, spermine and water; iriter-molecular contacts and base-stacking; flexibility of the molecule; conformational analysis of nucleotides in the structure. Among the more noteworthy features are a considerable irregularity in the helicity of the base-paired stems, a greater flexibility in the anticodon and aminoacyl acceptor arms, and a “coupling” among several conformational angles. The functional implications of these structural features are also discussed.     

Crystal structure analysis and refinement at 2.5 A of hexameric C-phycocyanin from the cyanobacterium Agmenellum quadruplicatum. The molecular model and its implications for light-harvesting

The crystal structure of the light-harvesting protein-pigment complex C-phycocyanin from the cyanobacterium Agmenellum quadruplicatum has been determined by Patterson search techniques on the basis of the molecular model of C-phycocyanin from Mastigocladus laminosus. The crystal unit cell (space group P321) contains three (alpha beta)6 hexamers centred on the crystallographic triads. The hexamer at the origin of the unit cell exhibits crystallographic 32 point symmetry. The other two hexamers (independent of the former) show crystallographic 3-fold and local 2-fold symmetry. The 3-fold redundancy of the asymmetric unit of the crystal cell was used in the refinement process, which proceeded by cyclic averaging, model building and energy-restrained crystallographic refinement. Refinement was terminated with a conventional crystallographic R-value of 0.20 with data to 2.5 A resolution. The two independent hexamers of the unit cell are identical within the limits of error at all levels of aggregation. Two trimers, which closely resemble the M. laminosus C-phycocyanin, are aggregated head-to-head to form the hexamer. Both trimers fit complementarily and are held together by polar and ionic interactions. Conservation of the amino acid residues involved in protein-chromophore and intermonomer interactions suggests common structural features for all biliproteins. Most probably, the hexameric aggregation form present in the crystals is closely related to the discs of native phycobilisome rods. All tetrapyrrole chromophores are extended but with different geometries enforced by different protein surroundings. In particular, interactions of the propionic side-chains with arginine residues and of the pyrrole nitrogen atoms with aspartate residues define configuration and conformation of the chromophores. Relative chromophore distances and orientations have been determined and a preferential pathway for the energy transfer suggested. Accordingly, within a hexamer the absorbed energy is funneled to chromophore B84 and then transduced via B84 chromophores along the phycobilisome rods.     

Refined structure of baboon alpha-lactalbumin at 1.7 A resolution. Comparison with C-type lysozyme

The solution of the structure of alpha-lactalbumin from baboon milk (Papio cynocephalus) at 4.5 A resolution using the isomorphous replacement method has been reported previously. Initial refinement on the basis of these low-resolution studies was not successful because of the poor isomorphism of the best heavy-atom derivative. Because of the striking similarity between the structure of lysozyme and alpha-lactalbumin, a more cautious molecular replacement approach was tried to refine the model. Using hen egg-white lysozyme as the starting model, preliminary refinement was performed using heavily constrained least-squares minimization in reciprocal space. The model was further refined using stereochemical restraints at 1.7 A resolution to a conventional crystallographic residual of 0.22 for 1141 protein atoms. In the final model, the root-mean-square deviation from ideality for bond distances is 0.015 A, and for angle distances it is 0.027 A. The refinement was carried out using the human alpha-lactalbumin sequence and "omit maps" calculated during the course of refinement indicated eight possible sequence changes in the baboon alpha-lactalbumin X-ray sequence. During the refinement, a tightly bound calcium ion and 150 water molecules, of which four are internal, have been located. Some of the water molecules were modelled for disordered side-chains. The co-ordination around the calcium is a slightly distorted pentagonal bipyramid. The Ca-O distances vary from 2.2 A to 2.6 A, representing a tight calcium-binding loop in the structure. The calcium-binding fold only superficially resembles the "EF-hand" and presumably has no evolutionary relationship with other EF-hand structures. The overall structure of alpha-lactalbumin is very similar to that of lysozyme. All large deviations occur in the loops where all sequence deletions and insertions are found. The C terminus appears to be rather flexible in alpha-lactalbumin compared to lysozyme. The experimental evidence supports the earlier predictions for the alpha-lactalbumin structure that were based upon the assumption that alpha-lactalbumin and lysozyme have similar three-dimensional structures, with minimal deletions and insertions. A detailed comparison of the two structures shows striking features as well as throwing some light on the evolution of these two proteins from a common precursor.     

Crystallographic Refinement and Structure-Factor Time-Averaging by Molecular Dynamics in the Absence of a Physical Force Field

Abstract

The application of Molecular-Dynamics simulation in protein-crystallographic structure refinement has become common practice. In this paper, structure optimizations are described where the driving force is derived only from the crystallographic data and not from any physical potential energy function. Under this extreme condition ab initio structure refinement and the application of structure-factor time averaging was investigated using a small 9 atom test system. Success in ab initio refinement, where the starting atomic positions are randomly distributed, depends on the resolution of the crystallographic data used in the optimization. The presence of high resolution data introduces false minima in the X-ray energy profile, enhancing the search problem significantly. On the same system, we also tested the method of time-averaged crystallographically restrained Molecular Dynamics, again in the absence of a physical force field. In this method, the diffraction data is modelled by an ensemble of structures instead of one single structure. In comparison to conventional single-structure refinement, more reflections were required to determine a correct atomic distribution. A time-averaging simulation at 0.2 nm resolution (40 reflections) yielded an incorrect distribution, although a low R-factor was obtained. Simulations at 0.1 nm resolution (248 reflections) gave both low R-factors, 3 to 4%, and correct atomic distributions. The scale factor between the observed and time-averaged calculated structure factor amplitudes appeared to be unstable, when optimized during a time-averaging simulation. Tests of time-averaged restrained simulations with noise added to the observed structure-factor amplitudes, indicated that noise is modelled when no information in the form of constraints or restraints is available to distinguish it from real data.