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Michael Nilges

Institut Pasteur de Paris, Structural Biology and Chemistry, Faculty Member

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Papers by Michael Nilges

Three-dimensional structure of potato carboxypeptidase inhibitor in solution. A study using nuclear magnetic resonance, distance geometry, and restrained molecular dynamics

Biochemistry, 1987

The solution conformation of potato carboxypeptidase inhibitor (CPI) has been investigated by 'H ... more The solution conformation of potato carboxypeptidase inhibitor (CPI) has been investigated by 'H N M R spectroscopy. The spectrum is assigned in a sequential manner by using two-dimensional N M R techniques to identify through-bond and through-space (C5 A) connectivities. A set of 309 approximate interproton distance restraints is derived from the two-dimensional nuclear Overhauser enhancement spectra and used as the basis of a three-dimensional structure determination by a combination of metric matrix distance geometry and restrained molecular dynamics calculations. A total of 1 1 converged distance geometry structures were computed and refined by using restrained molecular dynamics. The average atomic root mean square (rms) difference between the final 11 structures and the mean structure obtained by averaging their coordinates is 1.4 f 0.3 8, for residues 2-39 and 0.9 f 0.2 %, for residues 5-37. The corresponding values for all atoms are 1.9 f 0.3 and 1.4 f 0.2 A, respectively. The larger values for residues 2-38 relative to those for residues 5-37 arise from the fact that the positions of the N -(residues 1-4) and C-(residues 38-39) terminal tails are rather poorly determined, whereas those of the core of the protein (residues 5-37) are well determined by the experimental interproton distance data. The computed structures are very close to the X-ray structure of CPI in its complex with carboxypeptidase, and the backbone atomic rms difference between the mean of the computed structures and the X-ray structure is only 1.2 A. Nevertheless, there are some real differences present which are evidenced by significant deviations between the experimental upper interproton distance limits and the corresponding interproton distances derived from the X-ray structure. These principally occur in two regions, residues 18-20 and residues 28-30, the latter comprising part of the region of secondary contacts between CPI and carboxypeptidase in the X-ray structure.

Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

Biochemistry, 1989

The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose ... more The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose binding domain of cellobiohydrolase I (CT-CBH I) from Trichoderma reesei was investigated by nuclear magnetic resonance ( N M R ) spectroscopy. The 'H N M R spectrum was completely assigned in a sequential manner by two-dimensional N M R techniques. A large number of stereospecific assignments for P-methylene protons, as well as ranges for the 4, J/, and x1 torsion angles, were obtained on the basis of sequential and intraresidue nuclear Overhauser enhancement (NOE) and coupling constant data in combination with a conformational data base search. The structure calculations were carried out in an iterative manner by using the hybrid distance geometry-dynamical simulated annealing method. This involved computing a series of initial structures from a subset of the experimental data in order to resolve ambiguities in the assignments of some N O E cross-peaks arising from chemical shift degeneracy. Additionally, this permitted us to extend the stereospecific assignments to the a-methylene protons of glycine using information on 4 torsion angles derived from the initial structure calculations. The final experimental data set consisted of 554 interproton distance restraints, 24 restraints for 12 hydrogen bonds, and 33 4, 24 J/, and 25 xI torsion angle restraints. CT-CBH I has two disulfide bridges whose pairing was previously unknown. Analysis of structures calculated with all three possible combinations of disulfide bonds, as well as without disulfide bonds, indicated that the correct disulfide bridge pairing was 8-25 and 19-35. Forty-one structures were computed with the 8-25 and 19-35 disulfide bridges, and the average atomic rms difference between the individual structures and the mean structure obtained by averaging their coordinates was 0.33 f 0.04 8, for the backbone atoms and 0.52 f 0.06 8, for all atoms. The protein has a wedgelike shape with an amphiphilic character, one face being predominantly hydrophilic and the other mainly hydrophobic. The principal element of secondary structure is made up of an irregular triple-stranded antiparallel @-sheet composed of residues 5-9 (PI), 24-28 (P2), and 33-36 (P3) in which strand P3 is hydrogen bonded to the other two strands. Strand Pl is preceded by and overlaps with a type I1 turn (residues 3-6), and strands P2 and P3 are connected by a type I turn (residues 29-32). Strands PI and 02 are connected by a loop (residues 14-19) via type I1 turns (residues 10-13 and 20-23). A number of side-chain interactions are discussed in the light of the structure.

Determination of three-dimensional structures of proteins from interproton distance data by hybrid distance geometry-dynamical simulated annealing calculations

Febs Letters, 1988

A new hybrid distance space-real space method for determining three-dimensional structures of pro... more A new hybrid distance space-real space method for determining three-dimensional structures of proteins on the basis of interproton distance restraints is presented. It involves the following steps: (i) the approximate polypeptide fold is obtained by generating a set of substructures comprising only a small subset of atoms by projection from multi-dimensional distance space into three-dimensional Cartesian coordinate space using a procedure known as 'embedding'; (ii) all remaining atoms are then added by best fitting extended amino acids one residue at a time to the substructures; (iii) the resulting structures are used as the starting point for real space dynamical simulated annealing calculations. The latter involve heating the system to a high temperature followed by slow cooling in order to overcome potential barriers along the pathway towards the global minimum region. This is carried out by solving Newton's equations of motion. Unlike conventional restrained molecular dynamics, however, the non-bonded interactions are represented by a simple van der Waals repulsion term. The method is illustrated by calculations on crambin (46 residues) and the globular domain of histone H5 (79 residues). It is shown that the hybrid method is more efficient computationally and samples a larger region of conformational space consistent with the experimental data than full metric matrix distance geometry calculations alone, particularly for large systems.

Computational challenges for macromolecular structure determination by X-ray crystallography and solution NMRspectroscopy

Quarterly Reviews of Biophysics, 1993

Page 1. Quarterly Reviews of Biophysics 26, 1 (1993), pp. 49-125 49 Printed in Great Britain Comp... more

Three-dimensional structure of phoratoxin in solution: combined use of nuclear magnetic resonance, distance geometry, and restrained molecular dynamics

Biochemistry, 1987

The solution conformation of phoratoxin, a 46-residue plant protein, has been investigated by 'H ... more The solution conformation of phoratoxin, a 46-residue plant protein, has been investigated by 'H nuclear magnetic resonance ( N M R ) spectroscopy. The spectrum is assigned in a sequential manner by a combination of two-dimensional N M R techniques to demonstrate through-bond and through-space (<5 A) connectivities. A set of 331 approximate interproton distance restraints and six 4 backbone torsion angle restraints is derived from the two-dimensional nuclear Overhauser enhancement and double quantum filtered homonuclear correlated spectra, respectively. These restraints are used as the basis of a structure determination with a metric matrix distance geometry algorithm. A total of eight structures are computed in this manner and subjected to refinement by restrained molecular dynamics in which the experimental restraints are incorporated into the total energy function of the system in the form of square well effective potentials. The average atomic root mean square difference between the final eight structures and the mean structure obtained by averaging their coordinates is 1.7 f 0.5 8, for the backbone atoms and 2.1 f 0.5 8, for all atoms. The overall shape of phoratoxin is that of the capital letter L, similar to that of crambin and a,-purothionin, with the longer arm comprising two a-helices at an angle of -140° to each other and

Determination of three-dimensional structures of proteins from interproton distance data by dynamical simulated annealing from a random array of atoms Circumventing problems associated with folding

Febs Letters, 1988

A new hybrid distance space-real space method for determining three-dimensional structures of pro... more A new hybrid distance space-real space method for determining three-dimensional structures of proteins on the basis of interproton distance restraints is presented. It involves the following steps: (i) the approximate polypeptide fold is obtained by generating a set of substructures comprising only a small subset of atoms by projection from multi-dimensional distance space into three-dimensional Cartesian coordinate space using a procedure known as 'embedding'; (ii) all remaining atoms are then added by best fitting extended amino acids one residue at a time to the substructures; (iii) the resulting structures are used as the starting point for real space dynamical simulated annealing calculations. The latter involve heating the system to a high temperature followed by slow cooling in order to overcome potential barriers along the pathway towards the global minimum region. This is carried out by solving Newton's equations of motion. Unlike conventional restrained molecular dynamics, however, the non-bonded interactions are represented by a simple van der Waals repulsion term. The method is illustrated by calculations on crambin (46 residues) and the globular domain of histone H5 (79 residues). It is shown that the hybrid method is more efficient computationally and samples a larger region of conformational space consistent with the experimental data than full metric matrix distance geometry calculations alone, particularly for large systems.

Crystallography and NMR system: a new software suite for macromolecular structure determina-tion

Determination of three-dimensional structures of proteins by simulated annealing with interproton distance restraints. Application to crambin, potato carboxypeptidase inhibitor and barley serine proteinase inhibitor 2

Protein Engineering Design & Selection, 1988

A new hybrid distance space-real space method for determining three-dimensional structures of pro... more A new hybrid distance space-real space method for determining three-dimensional structures of proteins on the basis of interproton distance restraints is presented. It involves the following steps: (i) the approximate polypeptide fold is obtained by generating a set of substructures comprising only a small subset of atoms by projection from multi-dimensional distance space into three-dimensional Cartesian coordinate space using a procedure known as 'embedding'; (ii) all remaining atoms are then added by best fitting extended amino acids one residue at a time to the substructures; (iii) the resulting structures are used as the starting point for real space dynamical simulated annealing calculations. The latter involve heating the system to a high temperature followed by slow cooling in order to overcome potential barriers along the pathway towards the global minimum region. This is carried out by solving Newton's equations of motion. Unlike conventional restrained molecular dynamics, however, the non-bonded interactions are represented by a simple van der Waals repulsion term. The method is illustrated by calculations on crambin (46 residues) and the globular domain of histone H5 (79 residues). It is shown that the hybrid method is more efficient computationally and samples a larger region of conformational space consistent with the experimental data than full metric matrix distance geometry calculations alone, particularly for large systems.

Crystallography & NMR System: A New Software Suite for Macromolecular Structure Determination

Acta Crystallographica Section D-biological Crystallography, 1998

A new software suite, called Crystallography & NMR System (CNS), has been developed for macromole... more A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible.

Sampling and efficiency of metric matrix distance geometry: A novel partial metrization algorithm

Journal of Biomolecular Nmr, 1992

In this paper, we present a reassessment of the sampling properties of the metric matrix distance... more In this paper, we present a reassessment of the sampling properties of the metric matrix distance geometry algorithm, which is in wide-spread use in the determination of three-dimensional structures from nuclear magnetic resonance (NMR) data. To this end, we compare the conformational space sampled by structures generated with a variety of metric matrix distance geometry protocols. As test systems we use an unconstrained polypeptide, and a small protein (rabbit neutrophil defensin peptide 5) for which only few tertiary distances had been derived from the NMR data, allowing several possible folds of the polypeptide chain. A process called ‘metrization’ in the preparation of a trial distance matrix has a very large effect on the sampling properties of the algorithm. It is shown that, depending on the metrization protocol used, metric matrix distance geometry can have very good sampling properties'indeed, both for the unconstrained model system and the NMR-structure case. We show that the sampling properties are to a great degree determined by the way in which the first few distances are chosen within their bounds. Further, we present a new protocol (‘partial metrization’) that is computationally more efficient but has the same excellent sampling properties. This novel protocol has been implemented in an expanded new release of the program X-PLOR with distance geometry capabilities.

Internal repeats in the BRCA2 protein sequence

Nature Genetics, 1996

Structure of the pleckstrin homology domain from beta-spectrin

Nature, 1994

The &amp;amp;#39;pleckstrin homology&amp;amp;#39; or PH domain is a 100-residue protein m... more The &amp;amp;#39;pleckstrin homology&amp;amp;#39; or PH domain is a 100-residue protein module. It is present in many kinases, different isoforms of phospholipase C, GTPase-activating proteins and nucleotide-exchange factors. Its function is not known, but many proteins that contain a PH domain interact with GTP-binding proteins. The PH domain in beta-adrenergic receptor kinase may be involved in binding to the beta gamma subunits of a trimeric G-protein. We report here the three-dimensional structure of the PH domain of the cytoskeletal protein spectrin using homonuclear nuclear magnetic resonance. The core of the molecule is an antiparallel beta-sheet consisting of seven strands. The C terminus is folded into a long alpha-helix, and another helix is present in one of the surface loops. The molecule is electrostatically polarized and contains a pocket which may be involved in the binding of a ligand. There is a distant relationship to the peptidyl-prolyl-cis-trans-isomerase FKBP in which this pocket is involved in the binding of the macrocyclic compound FK506 (refs 8-11).

The PH superfold: a structural scaffold for multiple functions

Trends in Biochemical Sciences, 1999

Pleckstrin homology (PH) domains form a structurally conserved family that is associated with man... more Pleckstrin homology (PH) domains form a structurally conserved family that is associated with many regulatory pathways within the cell. Domains with a nearly identical fold are found in other families that share no sequence similarity, suggesting the existence of a stable PH superfold. The PH domains generally function as regulated membrane-binding modules that bind to inositol lipids and respond to upstream signals by targeting the host proteins to the correct cellular sites. The other domains with a similar fold, such as the phosphotyrosine binding domains, recognize protein ligands.

The folding catalyst protein disulfide isomerase is constructed of active and inactive thioredoxin modules

Current Biology, 1997

Background: Protein disulfide isomerase (PDI), a multifunctional protein of the endoplasmic retic... more Background: Protein disulfide isomerase (PDI), a multifunctional protein of the endoplasmic reticulum, catalyzes the formation, breakage and rearrangement of disulfide bonds during protein folding. Dissection of this protein into its individual domains has confirmed the presence of the a and a′ domains, which are homologous to thioredoxin, having related structures and activities. The a and a′ domains both contain a –Cys–Gly–His–Cys– active-site sequence motif. The remainder of the molecule consists primarily of two further domains, designated b and b′ which are thought to be sequence repeats on the basis of a limited sequence similarity. The functions of the b and b′ domains are unknown and, until now, the structure of neither domain was known.Results: Heteronuclear nuclear magnetic resonance (NMR) methods have been used to determine the global fold of the PDI b domain. The protein has an α/β fold with the order of the elements of secondary structure being β1–α1–β2–α2–β3–α3–β4–β5–α4. The strands are all in a parallel arrangement with respect to each other, except for β4 which is antiparallel. The arrangement of the secondary structure elements of the b domain is identical to that found in the a domain of PDI and in the ubiquitous redox protein thioredoxin; the three-dimensional folding topology of the b domain is also very similar to that of these proteins.Conclusions: Our determination of the global fold of the b domain of PDI by NMR reveals that, like the a domain, the b domain contains the thioredoxin motif, even though the b domain has no significant amino-acid sequence similarities to any members of the thioredoxin family. This observation, together with indications that the b′ domain adopts a similar fold, suggests that PDI consists of active and inactive thioredoxin modules. These modules may have been adapted during evolution to provide PDI with its complete spectrum of enzymatic activities.

Structure Determination of the N-Terminal Thioredoxin-like Domain of Protein Disulfide Isomerase Using Multidimensional Heteronuclear 13 C/ 15 N NMR Spectroscopy

Biochemistry, 1996

As a first step in dissecting the structure of human protein disulfide isomerase (PDI), the struc... more As a first step in dissecting the structure of human protein disulfide isomerase (PDI), the structure of a fragment corresponding to the first 120 residues of its sequence has been determined using heteronuclear multidimensional NMR techniques. As expected from its primary structure homology, the fragment has the thioredoxin fold. Similarities and differences in their structures help to explain why thioredoxins are reductants, whereas PDI is an oxidant of protein thiol groups. The results confirm that PDI has a modular, multidomain structure, which will facilitate its structural and functional characterization.

Three-dimensional structure of potato carboxypeptidase inhibitor in solution. A study using nuclear magnetic resonance, distance geometry, and restrained molecular dynamics

Biochemistry, 1987

The solution conformation of potato carboxypeptidase inhibitor (CPI) has been investigated by 'H ... more The solution conformation of potato carboxypeptidase inhibitor (CPI) has been investigated by 'H N M R spectroscopy. The spectrum is assigned in a sequential manner by using two-dimensional N M R techniques to identify through-bond and through-space (C5 A) connectivities. A set of 309 approximate interproton distance restraints is derived from the two-dimensional nuclear Overhauser enhancement spectra and used as the basis of a three-dimensional structure determination by a combination of metric matrix distance geometry and restrained molecular dynamics calculations. A total of 1 1 converged distance geometry structures were computed and refined by using restrained molecular dynamics. The average atomic root mean square (rms) difference between the final 11 structures and the mean structure obtained by averaging their coordinates is 1.4 f 0.3 8, for residues 2-39 and 0.9 f 0.2 %, for residues 5-37. The corresponding values for all atoms are 1.9 f 0.3 and 1.4 f 0.2 A, respectively. The larger values for residues 2-38 relative to those for residues 5-37 arise from the fact that the positions of the N -(residues 1-4) and C-(residues 38-39) terminal tails are rather poorly determined, whereas those of the core of the protein (residues 5-37) are well determined by the experimental interproton distance data. The computed structures are very close to the X-ray structure of CPI in its complex with carboxypeptidase, and the backbone atomic rms difference between the mean of the computed structures and the X-ray structure is only 1.2 A. Nevertheless, there are some real differences present which are evidenced by significant deviations between the experimental upper interproton distance limits and the corresponding interproton distances derived from the X-ray structure. These principally occur in two regions, residues 18-20 and residues 28-30, the latter comprising part of the region of secondary contacts between CPI and carboxypeptidase in the X-ray structure.

Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

Biochemistry, 1989

The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose ... more The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose binding domain of cellobiohydrolase I (CT-CBH I) from Trichoderma reesei was investigated by nuclear magnetic resonance ( N M R ) spectroscopy. The 'H N M R spectrum was completely assigned in a sequential manner by two-dimensional N M R techniques. A large number of stereospecific assignments for P-methylene protons, as well as ranges for the 4, J/, and x1 torsion angles, were obtained on the basis of sequential and intraresidue nuclear Overhauser enhancement (NOE) and coupling constant data in combination with a conformational data base search. The structure calculations were carried out in an iterative manner by using the hybrid distance geometry-dynamical simulated annealing method. This involved computing a series of initial structures from a subset of the experimental data in order to resolve ambiguities in the assignments of some N O E cross-peaks arising from chemical shift degeneracy. Additionally, this permitted us to extend the stereospecific assignments to the a-methylene protons of glycine using information on 4 torsion angles derived from the initial structure calculations. The final experimental data set consisted of 554 interproton distance restraints, 24 restraints for 12 hydrogen bonds, and 33 4, 24 J/, and 25 xI torsion angle restraints. CT-CBH I has two disulfide bridges whose pairing was previously unknown. Analysis of structures calculated with all three possible combinations of disulfide bonds, as well as without disulfide bonds, indicated that the correct disulfide bridge pairing was 8-25 and 19-35. Forty-one structures were computed with the 8-25 and 19-35 disulfide bridges, and the average atomic rms difference between the individual structures and the mean structure obtained by averaging their coordinates was 0.33 f 0.04 8, for the backbone atoms and 0.52 f 0.06 8, for all atoms. The protein has a wedgelike shape with an amphiphilic character, one face being predominantly hydrophilic and the other mainly hydrophobic. The principal element of secondary structure is made up of an irregular triple-stranded antiparallel @-sheet composed of residues 5-9 (PI), 24-28 (P2), and 33-36 (P3) in which strand P3 is hydrogen bonded to the other two strands. Strand Pl is preceded by and overlaps with a type I1 turn (residues 3-6), and strands P2 and P3 are connected by a type I turn (residues 29-32). Strands PI and 02 are connected by a loop (residues 14-19) via type I1 turns (residues 10-13 and 20-23). A number of side-chain interactions are discussed in the light of the structure.

Determination of three-dimensional structures of proteins from interproton distance data by hybrid distance geometry-dynamical simulated annealing calculations

Febs Letters, 1988

A new hybrid distance space-real space method for determining three-dimensional structures of pro... more A new hybrid distance space-real space method for determining three-dimensional structures of proteins on the basis of interproton distance restraints is presented. It involves the following steps: (i) the approximate polypeptide fold is obtained by generating a set of substructures comprising only a small subset of atoms by projection from multi-dimensional distance space into three-dimensional Cartesian coordinate space using a procedure known as 'embedding'; (ii) all remaining atoms are then added by best fitting extended amino acids one residue at a time to the substructures; (iii) the resulting structures are used as the starting point for real space dynamical simulated annealing calculations. The latter involve heating the system to a high temperature followed by slow cooling in order to overcome potential barriers along the pathway towards the global minimum region. This is carried out by solving Newton's equations of motion. Unlike conventional restrained molecular dynamics, however, the non-bonded interactions are represented by a simple van der Waals repulsion term. The method is illustrated by calculations on crambin (46 residues) and the globular domain of histone H5 (79 residues). It is shown that the hybrid method is more efficient computationally and samples a larger region of conformational space consistent with the experimental data than full metric matrix distance geometry calculations alone, particularly for large systems.

Computational challenges for macromolecular structure determination by X-ray crystallography and solution NMRspectroscopy

Quarterly Reviews of Biophysics, 1993

Page 1. Quarterly Reviews of Biophysics 26, 1 (1993), pp. 49-125 49 Printed in Great Britain Comp... more

Three-dimensional structure of phoratoxin in solution: combined use of nuclear magnetic resonance, distance geometry, and restrained molecular dynamics

Biochemistry, 1987

The solution conformation of phoratoxin, a 46-residue plant protein, has been investigated by 'H ... more The solution conformation of phoratoxin, a 46-residue plant protein, has been investigated by 'H nuclear magnetic resonance ( N M R ) spectroscopy. The spectrum is assigned in a sequential manner by a combination of two-dimensional N M R techniques to demonstrate through-bond and through-space (<5 A) connectivities. A set of 331 approximate interproton distance restraints and six 4 backbone torsion angle restraints is derived from the two-dimensional nuclear Overhauser enhancement and double quantum filtered homonuclear correlated spectra, respectively. These restraints are used as the basis of a structure determination with a metric matrix distance geometry algorithm. A total of eight structures are computed in this manner and subjected to refinement by restrained molecular dynamics in which the experimental restraints are incorporated into the total energy function of the system in the form of square well effective potentials. The average atomic root mean square difference between the final eight structures and the mean structure obtained by averaging their coordinates is 1.7 f 0.5 8, for the backbone atoms and 2.1 f 0.5 8, for all atoms. The overall shape of phoratoxin is that of the capital letter L, similar to that of crambin and a,-purothionin, with the longer arm comprising two a-helices at an angle of -140° to each other and

Determination of three-dimensional structures of proteins from interproton distance data by dynamical simulated annealing from a random array of atoms Circumventing problems associated with folding

Febs Letters, 1988

A new hybrid distance space-real space method for determining three-dimensional structures of pro... more A new hybrid distance space-real space method for determining three-dimensional structures of proteins on the basis of interproton distance restraints is presented. It involves the following steps: (i) the approximate polypeptide fold is obtained by generating a set of substructures comprising only a small subset of atoms by projection from multi-dimensional distance space into three-dimensional Cartesian coordinate space using a procedure known as 'embedding'; (ii) all remaining atoms are then added by best fitting extended amino acids one residue at a time to the substructures; (iii) the resulting structures are used as the starting point for real space dynamical simulated annealing calculations. The latter involve heating the system to a high temperature followed by slow cooling in order to overcome potential barriers along the pathway towards the global minimum region. This is carried out by solving Newton's equations of motion. Unlike conventional restrained molecular dynamics, however, the non-bonded interactions are represented by a simple van der Waals repulsion term. The method is illustrated by calculations on crambin (46 residues) and the globular domain of histone H5 (79 residues). It is shown that the hybrid method is more efficient computationally and samples a larger region of conformational space consistent with the experimental data than full metric matrix distance geometry calculations alone, particularly for large systems.

Crystallography and NMR system: a new software suite for macromolecular structure determina-tion

Determination of three-dimensional structures of proteins by simulated annealing with interproton distance restraints. Application to crambin, potato carboxypeptidase inhibitor and barley serine proteinase inhibitor 2

Protein Engineering Design & Selection, 1988

A new hybrid distance space-real space method for determining three-dimensional structures of pro... more A new hybrid distance space-real space method for determining three-dimensional structures of proteins on the basis of interproton distance restraints is presented. It involves the following steps: (i) the approximate polypeptide fold is obtained by generating a set of substructures comprising only a small subset of atoms by projection from multi-dimensional distance space into three-dimensional Cartesian coordinate space using a procedure known as 'embedding'; (ii) all remaining atoms are then added by best fitting extended amino acids one residue at a time to the substructures; (iii) the resulting structures are used as the starting point for real space dynamical simulated annealing calculations. The latter involve heating the system to a high temperature followed by slow cooling in order to overcome potential barriers along the pathway towards the global minimum region. This is carried out by solving Newton's equations of motion. Unlike conventional restrained molecular dynamics, however, the non-bonded interactions are represented by a simple van der Waals repulsion term. The method is illustrated by calculations on crambin (46 residues) and the globular domain of histone H5 (79 residues). It is shown that the hybrid method is more efficient computationally and samples a larger region of conformational space consistent with the experimental data than full metric matrix distance geometry calculations alone, particularly for large systems.

Crystallography & NMR System: A New Software Suite for Macromolecular Structure Determination

Acta Crystallographica Section D-biological Crystallography, 1998

A new software suite, called Crystallography & NMR System (CNS), has been developed for macromole... more A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible.

Sampling and efficiency of metric matrix distance geometry: A novel partial metrization algorithm

Journal of Biomolecular Nmr, 1992

In this paper, we present a reassessment of the sampling properties of the metric matrix distance... more In this paper, we present a reassessment of the sampling properties of the metric matrix distance geometry algorithm, which is in wide-spread use in the determination of three-dimensional structures from nuclear magnetic resonance (NMR) data. To this end, we compare the conformational space sampled by structures generated with a variety of metric matrix distance geometry protocols. As test systems we use an unconstrained polypeptide, and a small protein (rabbit neutrophil defensin peptide 5) for which only few tertiary distances had been derived from the NMR data, allowing several possible folds of the polypeptide chain. A process called ‘metrization’ in the preparation of a trial distance matrix has a very large effect on the sampling properties of the algorithm. It is shown that, depending on the metrization protocol used, metric matrix distance geometry can have very good sampling properties'indeed, both for the unconstrained model system and the NMR-structure case. We show that the sampling properties are to a great degree determined by the way in which the first few distances are chosen within their bounds. Further, we present a new protocol (‘partial metrization’) that is computationally more efficient but has the same excellent sampling properties. This novel protocol has been implemented in an expanded new release of the program X-PLOR with distance geometry capabilities.

Internal repeats in the BRCA2 protein sequence

Nature Genetics, 1996

Structure of the pleckstrin homology domain from beta-spectrin

Nature, 1994

The &amp;amp;#39;pleckstrin homology&amp;amp;#39; or PH domain is a 100-residue protein m... more The &amp;amp;#39;pleckstrin homology&amp;amp;#39; or PH domain is a 100-residue protein module. It is present in many kinases, different isoforms of phospholipase C, GTPase-activating proteins and nucleotide-exchange factors. Its function is not known, but many proteins that contain a PH domain interact with GTP-binding proteins. The PH domain in beta-adrenergic receptor kinase may be involved in binding to the beta gamma subunits of a trimeric G-protein. We report here the three-dimensional structure of the PH domain of the cytoskeletal protein spectrin using homonuclear nuclear magnetic resonance. The core of the molecule is an antiparallel beta-sheet consisting of seven strands. The C terminus is folded into a long alpha-helix, and another helix is present in one of the surface loops. The molecule is electrostatically polarized and contains a pocket which may be involved in the binding of a ligand. There is a distant relationship to the peptidyl-prolyl-cis-trans-isomerase FKBP in which this pocket is involved in the binding of the macrocyclic compound FK506 (refs 8-11).

The PH superfold: a structural scaffold for multiple functions

Trends in Biochemical Sciences, 1999

Pleckstrin homology (PH) domains form a structurally conserved family that is associated with man... more Pleckstrin homology (PH) domains form a structurally conserved family that is associated with many regulatory pathways within the cell. Domains with a nearly identical fold are found in other families that share no sequence similarity, suggesting the existence of a stable PH superfold. The PH domains generally function as regulated membrane-binding modules that bind to inositol lipids and respond to upstream signals by targeting the host proteins to the correct cellular sites. The other domains with a similar fold, such as the phosphotyrosine binding domains, recognize protein ligands.

The folding catalyst protein disulfide isomerase is constructed of active and inactive thioredoxin modules

Current Biology, 1997

Background: Protein disulfide isomerase (PDI), a multifunctional protein of the endoplasmic retic... more Background: Protein disulfide isomerase (PDI), a multifunctional protein of the endoplasmic reticulum, catalyzes the formation, breakage and rearrangement of disulfide bonds during protein folding. Dissection of this protein into its individual domains has confirmed the presence of the a and a′ domains, which are homologous to thioredoxin, having related structures and activities. The a and a′ domains both contain a –Cys–Gly–His–Cys– active-site sequence motif. The remainder of the molecule consists primarily of two further domains, designated b and b′ which are thought to be sequence repeats on the basis of a limited sequence similarity. The functions of the b and b′ domains are unknown and, until now, the structure of neither domain was known.Results: Heteronuclear nuclear magnetic resonance (NMR) methods have been used to determine the global fold of the PDI b domain. The protein has an α/β fold with the order of the elements of secondary structure being β1–α1–β2–α2–β3–α3–β4–β5–α4. The strands are all in a parallel arrangement with respect to each other, except for β4 which is antiparallel. The arrangement of the secondary structure elements of the b domain is identical to that found in the a domain of PDI and in the ubiquitous redox protein thioredoxin; the three-dimensional folding topology of the b domain is also very similar to that of these proteins.Conclusions: Our determination of the global fold of the b domain of PDI by NMR reveals that, like the a domain, the b domain contains the thioredoxin motif, even though the b domain has no significant amino-acid sequence similarities to any members of the thioredoxin family. This observation, together with indications that the b′ domain adopts a similar fold, suggests that PDI consists of active and inactive thioredoxin modules. These modules may have been adapted during evolution to provide PDI with its complete spectrum of enzymatic activities.

Structure Determination of the N-Terminal Thioredoxin-like Domain of Protein Disulfide Isomerase Using Multidimensional Heteronuclear 13 C/ 15 N NMR Spectroscopy

Biochemistry, 1996

As a first step in dissecting the structure of human protein disulfide isomerase (PDI), the struc... more As a first step in dissecting the structure of human protein disulfide isomerase (PDI), the structure of a fragment corresponding to the first 120 residues of its sequence has been determined using heteronuclear multidimensional NMR techniques. As expected from its primary structure homology, the fragment has the thioredoxin fold. Similarities and differences in their structures help to explain why thioredoxins are reductants, whereas PDI is an oxidant of protein thiol groups. The results confirm that PDI has a modular, multidomain structure, which will facilitate its structural and functional characterization.