PROTEINS Structure, Function, and Genetics 10:117-129 (1991)
Relaxation Data in NMR Structure Determination: Model Calculations for the Lysozyme-Gd3 Complex +
Michael J. Sutcliffe and Christopher M. Dobson Oxford Centre for Molecular Sciences and Znorpanic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, United Kingdom OX1 3QR
ABSTRACT The effect of including paramagnetic relaxation data as additional restraints in the determination of protein tertiary structures from NMR data has been explored by a systematic series of model calculations. The system used for testing the method was the 2.0 A resolution tetragonal crystal structure of hen egg white lysozyme (129 amino acid residues) and structures were generated using a version of the hybrid “distance geometry-dynamic simulated annealing” procedure. A limited set of 769 NOES was used as restraints in all the calculations; the strengths of these were categorized into three classes on the basis of distances observed in the crystal structure. The values of 50 angles were also restrained on the basis of amide-alpha coupling constants calculated from the X-ray structure. Five sets of 12 structures were determined using differing sets of paramagnetic relaxation data as restraints additional to those involving the NOE and coupling constant data. The paramagnetic relaxation data were modeled on the basis of the distances of defined protons from the crystallographic binding site of Gd3’ in lysozyme. Analysis of the results showed that the relaxation data significantly improved the correspondence between the set of generated structures and the crystal structure, and that the more well defined the relaxation data, the more significant the improvement in the quality of the structures. The results suggest that the inclusion of paramagnetic relaxation restraints could be of significant value for the experimental determination of protein structures from NMR data.
+
mined by NMR and, although most have been small (less than -60 residues) the structures of a number of larger proteins have been successfully defined. The use of a variety of heteronuclear techniques in particular (e.g., utilizing isotopic labelin$*4) in conjunction with multidimensional approaches (e.g., 3-D NMR5s6) promises to bring increasingly larger proteins within the scope of the method. Structure determination methods utilising NMR data can be divided into three steps. The first involves assignment of resonances to specific residues in the protein. The next step requires measurement of NMR parameters which are dependent upon the structure. These are conventionally NOEs7r8 which relate to interproton distances and spin-spin coupling constants’ which relate to specific dihedral angles. Finally, generation of the three-dimensional structure is carried out by searching for conformations consistent with the NMR restraints and the usual restrictions of chemical bonding.lo,l1 The level of precision in such structure determinations is generally strongly dependent on the number of NMR restraints which can be obtained per residue. The high precision structures of tendamistat12 and BDS-I (from sea anemone)13are derived from an average of almost 13 NMR restraints per residue. As larger protein molecules are studied, the number of restraints required increases and the task of acquiring the appropriate NOE and coupling constant data becomes increasingly difficult. It is therefore of interest to consider if there are additional sets of geometric restraints available which can be utilized in the determination of protein structures from solution NMR experiments. One possibility involves interpretation of the readily measured chemical shift values; these have been used extensively in comparing crystal and solution structures (e.g., Perkins14)
Key words: protein NMR, distance restraints, paramagnetic relaxation, protein structure determination INTRODUCTION Over recent years, nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful technique for the independent determination of protein tertiary structures in solution (see Wuthrich’ and Clore and Gronenborn2 for reviews). A substantial number of protein structures has now been deter0 1991 WILEY-LISS, INC.
Received July 17, 1990; revision accepted September 28, 1990. Address reprint requests to Christopher M. Dobson, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, United Kingdom OX1 3QR. Present address of M.J. Sutcliffe: Biological NMR Centre, P.O. Box 138, Medical Sciences Building, Leicester University, University Road, Leicester, UK LE1 9HN.
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and could potentially be used as an additional set of restraints in structure determination. Chemical shifts, however, are not well understood and reflect the net result of a large number of interactions, unlike the pairwise interactions sampled by NOE and coupling constant measurements. Another possibility involves the use of paramagnetic effects such as those consequent upon binding of metal ions or attachment of spin labels. These methods have also been used to compare crystal and solution structures and have long been recognized as having the potential to provide independent structural information about macromolecules.15-18 Paramagnetic ions are in fact highly attractive as structural probes. Because of the large magnetic moment of the electron compared to the nuclei, large pertubations to NMR parameters are induced by unpaired electrons and effects on a wide range of nuclei, even those distant from the paramagnetic center, can be measured experimentally. The relationship of experimentally determined NMR pertubations to structural data is also well understood. The induced paramagnetic relaxation, for example, has a simple rP6dependence, like NOE effects; however, unlike with NOEs, spin diffusion effects do not complicate the interpretation of the data. In the case of induced paramagnetic shifts, an angular as well as a distance function is involved and additional information is therefore in principle available. Paramagnetic effects have not previously been used in NMR structure determination using the approach developed for utilizing NOE data. Prior to exploiting the results of experimental studies, we have explored model calculations to examine the possibile utility of this approach. In particular, we have assessed the value of including paramagnetic relaxation restraints alongside those from NOES and coupling constants. This was carried out by simulating a set of NOE and J-value restraints which were chosen to provide only a limited definition of the structure, and then exploring the influence of additional paramagnetic restraints.
METHODS Structure Determination Protocol The 2.0 A resolution crystal structure of lysozymelg was used to generate sets of model NMR restraints in a procedure similar to that used to explore the effectiveness of molecular dynamics refinement with crambin." The magnitudes of the NOES were modeled on the basis of proton-proton distances observed in the crystal structure as strong (
Relaxation Data in NMR Structure Determination: Model Calculations for the Lysozyme-Gd3 Complex +
Michael J. Sutcliffe and Christopher M. Dobson Oxford Centre for Molecular Sciences and Znorpanic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, United Kingdom OX1 3QR
ABSTRACT The effect of including paramagnetic relaxation data as additional restraints in the determination of protein tertiary structures from NMR data has been explored by a systematic series of model calculations. The system used for testing the method was the 2.0 A resolution tetragonal crystal structure of hen egg white lysozyme (129 amino acid residues) and structures were generated using a version of the hybrid “distance geometry-dynamic simulated annealing” procedure. A limited set of 769 NOES was used as restraints in all the calculations; the strengths of these were categorized into three classes on the basis of distances observed in the crystal structure. The values of 50 angles were also restrained on the basis of amide-alpha coupling constants calculated from the X-ray structure. Five sets of 12 structures were determined using differing sets of paramagnetic relaxation data as restraints additional to those involving the NOE and coupling constant data. The paramagnetic relaxation data were modeled on the basis of the distances of defined protons from the crystallographic binding site of Gd3’ in lysozyme. Analysis of the results showed that the relaxation data significantly improved the correspondence between the set of generated structures and the crystal structure, and that the more well defined the relaxation data, the more significant the improvement in the quality of the structures. The results suggest that the inclusion of paramagnetic relaxation restraints could be of significant value for the experimental determination of protein structures from NMR data.
+
mined by NMR and, although most have been small (less than -60 residues) the structures of a number of larger proteins have been successfully defined. The use of a variety of heteronuclear techniques in particular (e.g., utilizing isotopic labelin$*4) in conjunction with multidimensional approaches (e.g., 3-D NMR5s6) promises to bring increasingly larger proteins within the scope of the method. Structure determination methods utilising NMR data can be divided into three steps. The first involves assignment of resonances to specific residues in the protein. The next step requires measurement of NMR parameters which are dependent upon the structure. These are conventionally NOEs7r8 which relate to interproton distances and spin-spin coupling constants’ which relate to specific dihedral angles. Finally, generation of the three-dimensional structure is carried out by searching for conformations consistent with the NMR restraints and the usual restrictions of chemical bonding.lo,l1 The level of precision in such structure determinations is generally strongly dependent on the number of NMR restraints which can be obtained per residue. The high precision structures of tendamistat12 and BDS-I (from sea anemone)13are derived from an average of almost 13 NMR restraints per residue. As larger protein molecules are studied, the number of restraints required increases and the task of acquiring the appropriate NOE and coupling constant data becomes increasingly difficult. It is therefore of interest to consider if there are additional sets of geometric restraints available which can be utilized in the determination of protein structures from solution NMR experiments. One possibility involves interpretation of the readily measured chemical shift values; these have been used extensively in comparing crystal and solution structures (e.g., Perkins14)
Key words: protein NMR, distance restraints, paramagnetic relaxation, protein structure determination INTRODUCTION Over recent years, nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful technique for the independent determination of protein tertiary structures in solution (see Wuthrich’ and Clore and Gronenborn2 for reviews). A substantial number of protein structures has now been deter0 1991 WILEY-LISS, INC.
Received July 17, 1990; revision accepted September 28, 1990. Address reprint requests to Christopher M. Dobson, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, United Kingdom OX1 3QR. Present address of M.J. Sutcliffe: Biological NMR Centre, P.O. Box 138, Medical Sciences Building, Leicester University, University Road, Leicester, UK LE1 9HN.
118
M.J. SUTCLIFFE AND C.M. DOBSON
and could potentially be used as an additional set of restraints in structure determination. Chemical shifts, however, are not well understood and reflect the net result of a large number of interactions, unlike the pairwise interactions sampled by NOE and coupling constant measurements. Another possibility involves the use of paramagnetic effects such as those consequent upon binding of metal ions or attachment of spin labels. These methods have also been used to compare crystal and solution structures and have long been recognized as having the potential to provide independent structural information about macromolecules.15-18 Paramagnetic ions are in fact highly attractive as structural probes. Because of the large magnetic moment of the electron compared to the nuclei, large pertubations to NMR parameters are induced by unpaired electrons and effects on a wide range of nuclei, even those distant from the paramagnetic center, can be measured experimentally. The relationship of experimentally determined NMR pertubations to structural data is also well understood. The induced paramagnetic relaxation, for example, has a simple rP6dependence, like NOE effects; however, unlike with NOEs, spin diffusion effects do not complicate the interpretation of the data. In the case of induced paramagnetic shifts, an angular as well as a distance function is involved and additional information is therefore in principle available. Paramagnetic effects have not previously been used in NMR structure determination using the approach developed for utilizing NOE data. Prior to exploiting the results of experimental studies, we have explored model calculations to examine the possibile utility of this approach. In particular, we have assessed the value of including paramagnetic relaxation restraints alongside those from NOES and coupling constants. This was carried out by simulating a set of NOE and J-value restraints which were chosen to provide only a limited definition of the structure, and then exploring the influence of additional paramagnetic restraints.
METHODS Structure Determination Protocol The 2.0 A resolution crystal structure of lysozymelg was used to generate sets of model NMR restraints in a procedure similar to that used to explore the effectiveness of molecular dynamics refinement with crambin." The magnitudes of the NOES were modeled on the basis of proton-proton distances observed in the crystal structure as strong (
