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Comparison of NMR and crystal structures highlights conformational isomerism in protein active sites.

ABSTRACT: The JCSG has recently developed a protocol for systematic comparisons of high-quality crystal and NMR structures of proteins. In this paper, the extent to which this approach can provide function-related information on the two functionally annotated proteins TM1081, a Thermotoga maritima anti-? factor antagonist, and A2LD1 (gi:13879369), a mouse ?-glutamylamine cyclotransferase, is explored. The NMR structures of the two proteins have been determined in solution at 313 and 298?K, respectively, using the current JCSG protocol based on the software package UNIO for extensive automation. The corresponding crystal structures were solved by the JCSG at 100?K and 1.6?Å resolution and at 100?K and 1.9?Å resolution, respectively. The NMR and crystal structures of the two proteins share the same overall molecular architectures. However, the precision of the structure determination along the amino-acid sequence varies over a significantly wider range in the NMR structures than in the crystal structures. Thereby, in each of the two NMR structures about 65% of the residues have displacements below the average and in both proteins the less well ordered residues include large parts of the active sites, in addition to some highly solvent-exposed surface areas. Whereas the latter show increased disorder in the crystal and in solution, the active-site regions display increased displacements only in the NMR structures, where they undergo local conformational exchange on the millisecond time scale that appears to be frozen in the crystals. These observations suggest that a search for molecular regions showing increased structural disorder and slow dynamic processes in solution while being well ordered in the corresponding crystal structure might be a valid initial step in the challenge of identifying putative active sites in functionally unannotated proteins with known three-dimensional structure.

SUBMITTER: Serrano P

PROVIDER: S-EPMC2954230 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Comparison of NMR and crystal structures highlights conformational isomerism in protein active sites.

Serrano Pedro P Pedrini Bill B Geralt Michael M Jaudzems Kristaps K Mohanty Biswaranjan B Horst Reto R Herrmann Torsten T Elsliger Marc André MA Wilson Ian A IA Wüthrich Kurt K

Acta crystallographica. Section F, Structural biology and crystallization communications 20100930 Pt 10

The JCSG has recently developed a protocol for systematic comparisons of high-quality crystal and NMR structures of proteins. In this paper, the extent to which this approach can provide function-related information on the two functionally annotated proteins TM1081, a Thermotoga maritima anti-σ factor antagonist, and A2LD1 (gi:13879369), a mouse γ-glutamylamine cyclotransferase, is explored. The NMR structures of the two proteins have been determined in solution at 313 and 298 K, respectively, u ...[more]

PMID: 20944236

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Project description:In order for protein kinases to exchange nucleotide they must open and close their catalytic cleft. These motions are associated with rotations of the N-lobe, predominantly around the 'hinge region'. We conducted an analysis of 28 crystal structures of the serine-threonine kinase, p21-activated kinase 4 (PAK4), including three newly determined structures in complex with staurosporine, FRAX486, and fasudil (HA-1077). We find an unusual motion between the N-lobe and C-lobe of PAK4 that manifests as a partial unwinding of helix αC. Principal component analysis of the crystal structures rationalizes these movements into three major states, and analysis of the kinase hydrophobic spines indicates concerted movements that create an accessible back pocket cavity. The conformational changes that we observe for PAK4 differ from previous descriptions of kinase motions, and although we observe these differences in crystal structures there is the possibility that the movements observed may suggest a diversity of kinase conformational changes associated with regulation.Author summaryProtein kinases are key signaling proteins, and are important drug targets, therefore understanding their regulation is important for both basic research and clinical points of view. In this study, we observe unusual conformational 'hinging' for protein kinases. Hinging, the opening and closing of the kinase sub-domains to allow nucleotide binding and release, is critical for proper kinase regulation and for targeted drug discovery. We determine new crystal structures of PAK4, an important Rho-effector kinase, and conduct analyses of these and previously determined structures. We find that PAK4 crystal structures can be classified into specific conformational groups, and that these groups are associated with previously unobserved hinging motions and an unusual conformation for the kinase hydrophobic core. Our findings therefore indicate that there may be a diversity of kinase hinging motions, and that these may indicate different mechanisms of regulation.

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Comparison of NMR and crystal structures highlights conformational isomerism in protein active sites.

Publications

Comparison of NMR and crystal structures highlights conformational isomerism in protein active sites.

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