Johann Deisenhofer

The leucine-rich repeat: a versatile binding motif.

Leucine-rich repeats are short sequence motifs present in a number of proteins with diverse functions and cellular locations. All proteins containing these repeats are thought to be involved in protein-protein interactions. The crystal structure of ribonuclease inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual, nonglobular shape. These two features may be responsible for the protein-binding functions of proteins containing leucine-rich repeats.

Human α1-proteinase inhibitor: Crystal structure analysis of two crystal modifications, molecular model and preliminary analysis of the implications for function

Two closely related crystal structures of alpha 1-proteinase inhibitor modified at the reactive site peptide bond Met358--Ser359 have been analysed. The crystal structure has been obtained from diffraction data at 3 A resolution, with phases originally from isomorphous replacement. The electron density map was substantially improved by cyclic averaging of the electron densities of the two crystal forms and allowed the chain to be traced in terms of the known chemical amino acid sequence. Energy restrained crystallographic refinement was initiated and resulted in conventional R-values of 0.251 for the tetragonal crystal form (6 to 3 A resolution) and 0.247 for the hexagonal crystal form (6 to 3.2 A resolution). The polypeptide chain is almost completely arranged in well-defined secondary structural elements: three beta-sheets and eight alpha-helices. The helices are preferentially formed by the first 150 residues. They are in proximity underneath sheet A. The chain ends Met358 and Ser359 of the nicked species are arranged in strands on opposite ends of the molecule indicating a major structural rearrangement upon modification of the intact inhibitor. It is suggested that the Met358 strand is in a different conformation removed from sheet A and approaches Ser359 in the intact inhibitor species. Glu342, which is exchanged by a lysine in the Z-variant is in a strategic position for such a rearrangement. The three carbohydrate chains of alpha 1-proteinase inhibitor have partly defined electron density close to their attachment sites at asparagine residues. The anti-thrombin and ovalbumin amino acid sequences can be accommodated in the alpha 1 inhibitor molecular structure. The intron-exon junctions of the ovalbumin and the alpha 1-proteinase inhibitor gene are all in surface loops of the mature protein.

The Photosynthetic Reaction Center from the Purple Bacterium Rhodopseudomonas viridis

The history and methods of membrane protein crystallization are described. The solution of the structure of the photosynthetic reaction center from the bacterium Rhodopseudomonas viridis is described, and the structure of this membrane protein complex is correlated with its function as a light-driven electron pump across the photosynthetic membrane. Conclusions about the structure of the photosystem II reaction center from plants are drawn, and aspects of membrane protein structure are discussed.

Structure and function of cytochromes P450:a comparative analysis of three crystal structures

BACKGROUND Cytochromes P450 catalyze the oxidation of a variety of hydrophobic substrates. Sequence identities between P450 families are generally low (10-30%), and consequently, the structure-function correlations among P450s are not clear. The crystal structures of P450terp and the hemoprotein domain of P450BM-3 were recently determined, and are compared here with the previously available structure of P450cam. RESULTS The topology of all three enzymes is quite similar. The heme-binding core structure is well conserved, except for local differences in the I helices. The greatest variation is observed in the substrate-binding regions. The structural superposition of the proteins permits an improved sequence alignment of other P450s. The charge distribution in the three structures is similarly asymmetric and defines a molecular dipole. CONCLUSIONS Based on this comparison we believe that all P450s will be found to possess the same tertiary structure. The ability to precisely predict other P450 substrate-contact residues is limited by the extreme structural heterogeneity in the substrate-recognition regions. The central I-helix structures of P450terp and P450BM-3 suggest a role for helix-associated solvent molecules as a source of catalytic protons, distinct from the mechanism for P450cam. We suggest that the P450 molecular dipole might aid in both redox-partner docking and proton recruitment for catalysis.

Crystal structure of the outer membrane active transporter FepA from Escherichia coli.

Integral outer membrane receptors for iron chelates and vitamin B 12 carry out specific ligand transport against a concentration gradient. Energy for active transport is obtained from the proton–motive force of the inner membrane through physical interaction with TonB–ExbB–ExbD, an inner membrane complex. Here we report the crystal structure of an active transport, outer membrane receptor at 2.4 Å resolution. Two distinct functional domains are revealed: (i) a 22–stranded β–barrel that spans the outer membrane and contains large extracellular loops which appear to function in ligand binding; and (ii) a globular N–terminal domain that folds into the barrel pore, inhibiting access to the periplasm and contributing two additional loops for potential ligand binding. These loops could provide a signaling pathway between the processes of ligand recognition and TonB–mediated transport. The blockage of the pore suggests that the N–terminal domain must undergo a conformational rearrangement to allow ligand transport into the periplasm.

Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor: II. Crystallographic refinement at 1.9 Å resolution☆

Abstract The crystal structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor has been refined with data to 1.9 A resolution, using a procedure described by Deisenhofer & Steigemann (1974) in their refinement of the crystal structure of the free inhibitor. This procedure involves cycles consisting of phase calculation using the current atomic model, Fourier synthesis using these phases and the observed structure factor amplitudes and Diamonds real-space refinement (Diamond, 1971,1974). At various stages, difference Fourier syntheses are calculated to detect and correct gross errors in the model and to localize solvent molecules. The refinement progressed smoothly, starting with the model obtained from the isomorphous Fourier map at 2.6 A resolution. The R -factor is 0.23 for 20,500 significantly measured reflections to 1.9 A resolution, using an over-all temperature factor of 20 A 2 . The estimated standard deviation of atomic positions is 0.09 A. An objective assessment of the upper limit of the error in the atomic coordinates of the final model is possible by comparing the inhibitor component in the model of the complex with the refined structure of the free inhibitor (Deisenhofer & Steigemann, 1974). The mean deviation of main-chain atoms of the two molecular models in internal segments is 0.25 A, of main-chain dihedral angles 5.1 ° and side-chain dihedral angles 6.5 °. A comparison of the trypsin component with α-chymotrypsin (Birktoft & Blow, 1972) showed a mean deviation of main-chain atoms of 0.75 A. The structures are closely similar and the various deletions and insertions cause local structural differences only.

Pigment−protein interactions in the photosynthetic reaction centre from Rhodopseudomonas viridis

An X‐ray structure analysis of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis provides structural details of the pigment‐binding sites. The photosynthetic pigments are found in rather hydrophobic environments provided by the subunits L and M. In addition to apolar interactions, the bacteriochlorophylls of the primary electron donor (‘special pair’) and the bacteriopheophytins, but not the accessory bacteriochlorophylls, form hydrogen bonds with amino acid side chains of these protein subunits. The two branches of pigments which originate at the primary electron donor, and which mark possible electron pathways across the photosynthetic membrane, are in different environments and show different hydrogen bonding with the protein: this may help to understand why only one branch of pigments is active in the light‐driven electron transfer. The primary electron acceptor, a menaquinone (QA), is in a pocket formed by the M subunit and interacts with it by hydrophobic contacts and hydrogen bonds. Competitive inhibitors of the secondary quinone QB (o‐phenanthroline, the herbicide terbutryn) are bound into a pocket provided by the L subunit. Apart from numerous van der Waals interactions they also form hydrogen bonds to the protein.

Crystallographic refinement and atomic models of the intact immunoglobulin molecule Kol and its antigen-binding fragment at 3.0 Å and 1.9 Å resolution

Abstract The crystal structures of the intact immunoglobulin G1, (λ) Kol and its Fab † fragment were crystallographically refined at 3.0 A and 1.9 A resolution, respectively. The methods used were real space refinement (RLSP) energy and residual refinement (EREF), phase combination, constrained rigid body refinement (CORELS) and difference and Fourier map inspection. The final R -values are 0.24 and 0.26. These analyses allowed the construction of atomic models of parts not seen in detail in the previous analyses at 5 A and 3 A resolution, respectively (Colman et al. , 1976; Matsushima et al. , 1978): i.e. the hinge segment, the hypervariable segments and their intimate interaction with the hinge segment of a crystallographically related molecule. The hinge segment forms a short poly- l -proline double helix from Cys527 to Cys530 (Eu numbering 226 to 230). The preceding segment forms an open turn of helix. This segment and the segment following the poly- l -proline part, which was found to be flexible in Fc fragment crystals (Deisenhofer et al. , 1976) probably allow arm and stem movement of the antibody molecule. The combining site of Kol is compared with the combining site of Fab New (Saul et al. , 1978). The narrow cleft formed by the hypervariable loops in Kol is filled with aromatic amino acid side-chains. In the crystal, the hypervariable loops contact the hinge and adjacent segments of a related molecule accompanied by a substantial loss in accessible surface area. This contact is preserved in Kol Fab crystals and presumably occurs in the Kol cryoprecipitate. A comparison of the quaternary structures of intact Kol and Fab New showed, in addition to the large change in elbow angle (Colman et al. , 1976), changes in lateral domain association. These are discussed in the context of a possible signal transmission from the combining site to the distal end. An attempt was made to model build the IgG3 hinge segment, which is quadruplicated with respect to IgG1 (Michaelsen et al. , 1977), on the basis of the Kol hinge structure. A polyproline double helix appeared to be the most plausible model. The Fc part was found to be disordered in intact Kol crystals (Colman et al. , 1976). Refinement has reduced the electron density further in the crystal space, where the Fc parts must be located. Disorder, if static, must be fourfold or more in the crystalline state. Intensity measurements on Kol F(ab′) 2 and their comparison with intact Kol crystals provide evidence that the disorder is predominantly of a static nature.

Proteins with leucine-rich repeats

Leucine-rich repeats are short sequence motifs present in over sixty proteins, all of which appear to be involved in protein-protein interactions. The crystal structure of ribonuclease inhibitor demonstrated that the repeats correspond to beta-alpha structural units. The recently determined crystal structure of the ribonuclease A-ribonuclease inhibitor complex suggests the basis for the protein-binding function of leucine-rich repeats.

Structure of N-Terminal Domain of NPC1 Reveals Distinct Subdomains for Binding and Transfer of Cholesterol

LDL delivers cholesterol to lysosomes by receptor-mediated endocytosis. Exit of cholesterol from lysosomes requires two proteins, membrane-bound Niemann-Pick C1 (NPC1) and soluble NPC2. NPC2 binds cholesterol with its isooctyl side chain buried and its 3beta-hydroxyl exposed. Here, we describe high-resolution structures of the N-terminal domain (NTD) of NPC1 and complexes with cholesterol and 25-hydroxycholesterol. NPC1(NTD) binds cholesterol in an orientation opposite to NPC2: 3beta-hydroxyl buried and isooctyl side chain exposed. Cholesterol transfer from NPC2 to NPC1(NTD) requires reorientation of a helical subdomain in NPC1(NTD), enlarging the opening for cholesterol entry. NPC1 with point mutations in this subdomain (distinct from the binding subdomain) cannot accept cholesterol from NPC2 and cannot restore cholesterol exit from lysosomes in NPC1-deficient cells. We propose a working model wherein after lysosomal hydrolysis of LDL-cholesteryl esters, cholesterol binds NPC2, which transfers it to NPC1(NTD), reversing its orientation and allowing insertion of its isooctyl side chain into the outer lysosomal membranes.