LAHMA: structure analysis through local annotation of homology-matched amino acids.

Comparison of homologous structure models is a key step in analyzing protein structure. With a wealth of homologous structures, comparison becomes a tedious process, and often only a small (user-biased) selection of data is used. A multitude of structural superposition algorithms are then typically used to visualize the structures together in 3D and to compare them. Here, the Local Annotation of Homology-Matched Amino acids (LAHMA) website (https://lahma.pdb-redo.eu) is presented, which compares any structure model with all of its close homologs from the PDB-REDO databank. LAHMA displays structural features in sequence space, allowing users to uncover differences between homologous structure models that can be analyzed for their relevance to chemistry or biology. LAHMA visualizes numerous structural features, also allowing one-click comparison of structure-quality plots (for example the Ramachandran plot) and `in-browser' structural visualization of 3D models.

Interactions between N-terminal Modules in MPS1 Enable Spindle Checkpoint Silencing.

Faithful chromosome segregation relies on the ability of the spindle assembly checkpoint (SAC) to delay anaphase onset until chromosomes are attached to the mitotic spindle via their kinetochores. MPS1 kinase is recruited to kinetochores to initiate SAC signaling and is removed from kinetochores once stable microtubule attachments have been formed to allow normal mitotic progression. Here, we show that a helical fragment within the kinetochore-targeting N-terminal extension (NTE) module of MPS1 is required for interactions with kinetochores and forms intramolecular interactions with its adjacent tetratricopeptide repeat (TPR) domain. Bypassing this NTE-TPR interaction results in high MPS1 levels at kinetochores due to loss of regulatory input into MPS1 localization, inefficient MPS1 delocalization upon microtubule attachment, and SAC silencing defects. These results show that SAC responsiveness to attachments relies on regulated intramolecular interactions in MPS1 and highlight the sensitivity of mitosis to perturbations in the dynamics of the MPS1-NDC80-C interactions.

A crystal structure of the human protein kinase Mps1 reveals an ordered conformation of the activation loop.

Monopolar spindle 1 (Mps1) is a dual-specificity protein kinase, orchestrating faithful chromosome segregation during mitosis. All reported structures of the Mps1 kinase adopt the hallmarks of an inactive conformation, which includes a mostly disordered activation loop. Here, we present a 2.4 Å resolution crystal structure of an "extended" version of the Mps1 kinase domain, which shows an ordered activation loop. However, the other structural characteristics of an active kinase are not present. Our structure shows that the Mps1 activation loop can fit to the ATP binding pocket and interferes with ATP, but less so with inhibitors binding, partly explain the potency of various Mps1 inhibitors.

The Neuronal Tau Protein Blocks in Vitro Fibrillation of the Amyloid-ß (Aß) Peptide at the Oligomeric Stage.

In Alzheimer's disease, amyloid-ß (Aß) plaques and tau neurofibrillary tangles are the two pathological hallmarks. The co-occurrence and combined reciprocal pathological effects of Aß and tau protein aggregation have been observed in animal models of the disease. However, the molecular mechanism of their interaction remain unknown. Using a variety of biophysical measurements, we here show that the native full-length tau protein solubilizes the Aß40 peptide and prevents its fibrillation. The tau protein delays the amyloid fibrillation of the Aß40 peptide at substoichiometric ratios, showing different binding affinities toward the different stages of the aggregated Aß40 peptides. The Aß monomer structure remains random coil in the presence of tau, as observed by nuclear magnetic resonance (NMR), circular dichroism (CD) spectroscopy and photoinduced cross-linking methods. We propose a potential interaction mechanism for the influence of tau on Aß fibrillation.

Understanding inhibitor resistance in Mps1 kinase through novel biophysical assays and structures.

Monopolar spindle 1 (Mps1/TTK) is a protein kinase essential in mitotic checkpoint signaling, preventing anaphase until all chromosomes are properly attached to spindle microtubules. Mps1 has emerged as a potential target for cancer therapy, and a variety of compounds have been developed to inhibit its kinase activity. Mutations in the catalytic domain of Mps1 that give rise to inhibitor resistance, but retain catalytic activity and do not display cross-resistance to other Mps1 inhibitors, have been described. Here we characterize the interactions of two such mutants, Mps1 C604Y and C604W, which raise resistance to two closely related compounds, NMS-P715 and its derivative Cpd-5, but not to the well characterized Mps1 inhibitor, reversine. We show that estimates of the IC50 (employing a novel specific and efficient assay that utilizes a fluorescently labeled substrate) and the binding affinity (KD ) indicate that, in both mutants, Cpd-5 should be better tolerated than the closely related NMS-P715. To gain further insight, we determined the crystal structure of the Mps1 kinase mutants bound to Cpd-5 and NMS-P715 and compared the binding modes of Cpd-5, NMS-P715, and reversine. The difference in steric hindrance between Tyr/Trp604 and the trifluoromethoxy moiety of NMS-P715, the methoxy moiety of Cpd-5, and complete absence of such a group in reversine, account for differences we observe in vitro Our analysis enforces the notion that inhibitors targeting Mps1 drug-resistant mutations can emerge as a feasible intervention strategy based on existing scaffolds, if the clinical need arises.

Identification of productive and futile encounters in an electron transfer protein complex.

Well-defined, stereospecific states in protein complexes are often in exchange with an ensemble of more dynamic orientations: the encounter states. The structure of the stereospecific complex between cytochrome P450cam and putidaredoxin was solved recently by X-ray diffraction as well as paramagnetic NMR spectroscopy. Other than the stereospecific complex, the NMR data clearly show the presence of additional states in the complex in solution. In these encounter states, populated for a small percentage of the time, putidaredoxin assumes multiple orientations and samples a large part of the surface of cytochrome P450cam. To characterize the nature of the encounter states, an extensive paramagnetic NMR dataset has been analyzed using the Maximum Occurrence of Regions methodology. The analysis reveals the location and maximal spatial extent of the additional states needed to fully explain the NMR data. Under the assumption of sparsity of the size of the conformational ensemble, several minor states can be located quite precisely. The distribution of these minor states correlates with the electrostatic potential map around cytochrome P450cam. Whereas some minor states are on isolated positively charged patches, others are connected to the stereospecific site via positively charged paths. The existence of electrostatically favorable pathways between the stereospecific interaction site and the different minor states or lack thereof suggests a means to discriminate between productive and futile encounter states.

Structural basis of reversine selectivity in inhibiting Mps1 more potently than aurora B kinase.

Monopolar spindle 1 (Mps1, also known as TTK) is a protein kinase crucial for ensuring that cell division progresses to anaphase only after all chromosomes are connected to spindle microtubules. Incomplete chromosomal attachment leads to abnormal chromosome counts in the daughter cells (aneuploidy), a condition common in many solid cancers. Therefore Mps1 is an established target in cancer therapy. Mps1 kinase inhibitors include reversine (2-(4-morpholinoanilino)-6-cyclohexylaminopurine), a promiscuous compound first recognized as an inhibitor of the Aurora B mitotic kinase. Here, we present the 3.0-Å resolution crystal structure of the Mps1 kinase domain bound to reversine. Structural comparison of reversine bound to Mps1 and Aurora B, indicates a similar binding pose for the purine moiety of reversine making three conserved hydrogen bonds to the protein main chain, explaining the observed promiscuity of this inhibitor. The cyclohexyl and morpholinoaniline moieties of reversine however, have more extensive contacts with the protein in Mps1 than in Aurora B. This is reflected both in structure-based docking energy calculations, and in new experimental data we present here, that both confirm that the affinity of reversine towards Mps1 is about two orders of magnitude higher than towards Aurora B. Thus, our data provides detailed structural understanding of the existing literature that argues reversine inhibits Mps1 more efficiently than Aurora B based on biochemical and in-cell assays. Proteins 2016; 84:1761-1766. © 2016 Wiley Periodicals, Inc.

Delicate conformational balance of the redox enzyme cytochrome P450cam.

The energy landscapes of proteins are highly complex and can be influenced by changes in physical and chemical conditions under which the protein is studied. The redox enzyme cytochrome P450cam undergoes a multistep catalytic cycle wherein two electrons are transferred to the heme group and the enzyme visits several conformational states. Using paramagnetic NMR spectroscopy with a lanthanoid tag, we show that the enzyme bound to its redox partner, putidaredoxin, is in a closed state at ambient temperature in solution. This result contrasts with recent crystal structures of the complex, which suggest that the enzyme opens up when bound to its partner. The closed state supports a model of catalysis in which the substrate is locked in the active site pocket and the enzyme acts as an insulator for the reactive intermediates of the reaction.

CELL DIVISION CYCLE. Competition between MPS1 and microtubules at kinetochores regulates spindle checkpoint signaling.

Cell division progresses to anaphase only after all chromosomes are connected to spindle microtubules through kinetochores and the spindle assembly checkpoint (SAC) is satisfied. We show that the amino-terminal localization module of the SAC protein kinase MPS1 (monopolar spindle 1) directly interacts with the HEC1 (highly expressed in cancer 1) calponin homology domain in the NDC80 (nuclear division cycle 80) kinetochore complex in vitro, in a phosphorylation-dependent manner. Microtubule polymers disrupted this interaction. In cells, MPS1 binding to kinetochores or to ectopic NDC80 complexes was prevented by end-on microtubule attachment, independent of known kinetochore protein-removal mechanisms. Competition for kinetochore binding between SAC proteins and microtubules provides a direct and perhaps evolutionarily conserved way to detect a properly organized spindle ready for cell division.

Endogenous polyamines reduce the toxicity of soluble aß peptide aggregates associated with Alzheimer's disease.

Polyamines promote the formation of the Aß peptide amyloid fibers that are a hallmark of Alzheimer's disease. Here we show that polyamines interact with nonaggregated Aß peptides, thereby reducing the peptide's hydrophobic surface. We characterized the associated conformational change through NMR titrations and molecular dynamics simulations. We found that even low concentrations of spermine, sperimidine, and putrescine fully protected SH-SY5Y (a neuronal cell model) against the most toxic conformational species of Aß, even at an Aß oligomer concentration that would otherwise kill half of the cells or even more. These observations lead us to conclude that polyamines interfere with the more toxic prefibrillar conformations and might protect cells by promoting the structural transition of Aß toward its less toxic fibrillar state that we reported previously. Since polyamines are present in brain fluid at the concentrations where we observed all these effects, their activity needs to be taken into account in understanding the molecular processes related to the development of Alzheimer's disease.

Hot-spot residues in the cytochrome P450cam-putidaredoxin binding interface.

Cytochrome P450cam (P450cam) is a heme-containing monooxygenase that catalyzes the hydroxylation of D-camphor to produce 5-exo-hydroxycamphor. The catalytic cycle of P450cam requires two electrons, both of which are donated by putidaredoxin (Pdx), a ferredoxin containing a [2 Fe-2 S] cluster. Atomic-resolution structures of the Pdx-P450cam complex have recently been solved by X-ray crystallography and paramagnetic NMR spectroscopy. The binding interface showed the potential electron transfer pathways and interactions between Pdx Asp38 and P450cam Arg112, as well as hydrophobic contacts between the Pdx Trp106 and P450cam residues. Several polar residues not previously recognized as relevant for binding were found in the interface. In this study, site-directed mutagenesis, kinetic measurements, and NMR studies were employed to probe the energetic importance and role of the polar residues in the Pdx-P450cam interaction. A double mutant cycle (DMC) analysis of kinetic data shows that favorable interactions exist between Pdx Tyr33 and P450cam Asp125, as well as between Pdx Ser42 and P450cam His352. The results show that alanine substitutions of these residues and several others do not influence the rates of electron transfer. It is concluded that these polar interactions contribute to partner recognition rather than to electronic coupling of the redox centers.

The structure of the cytochrome p450cam-putidaredoxin complex determined by paramagnetic NMR spectroscopy and crystallography.

Cytochrome P450cam catalyzes the hydroxylation of camphor in a complex process involving two electron transfers (ETs) from the iron-sulfur protein putidaredoxin. The enzymatic control of the successive steps of catalysis is critical for a highly efficient reaction. The injection of the successive electrons is part of the control system. To understand the molecular interactions between putidaredoxin and cytochrome P450cam, we determined the structure of the complex both in solution and in the crystal state. Paramagnetic NMR spectroscopy using lanthanide tags yielded 446 structural restraints that were used to determine the solution structure. An ensemble of 10 structures with an RMSD of 1.3Å was obtained. The crystal structure of the complex was solved, showing a position of putidaredoxin that is identical with the one in the solution structure. The NMR data further demonstrate the presence of a minor state or set of states of the complex in solution, which is attributed to the presence of an encounter complex. The structure of the major state shows a small binding interface and a metal-to-metal distance of 16Å, with two pathways that provide strong electronic coupling of the redox centers. The interpretation of these results is discussed in the context of ET. The structure indicates that the ET rate can be much faster than the reported value, suggesting that the process may be gated.

A solution model of the complex formed by adrenodoxin and adrenodoxin reductase determined by paramagnetic NMR spectroscopy.

Lanthanide tags offer the opportunity to retrieve long-range distance information from NMR experiments that can be used to guide protein docking. To determine whether sufficient restraints can be retrieved for proteins with low solubility and availability, Ln tags were applied in the study of the 65 kDa membrane-associated protein complex formed by the electron carrier adrenodoxin and its electron donor, adrenodoxin reductase. The reductase is only monomeric at low concentration, and the paramagnetic iron-sulfur cluster of adrenodoxin broadens many of the resonances of nuclei in the interface. Guided by the paramagnetic restraints obtained using two Ln-tag attachment sites, protein docking yields a cluster of solutions with an rmsd of 3.2 A. The mean structure is close to the crystal structure of the cross-linked complex, with an rmsd of 4.0 A. It is concluded that with the application of Ln tags paramagnetic NMR restraints for structure determination can be retrieved even for difficult, low-concentration protein complexes.

Validation of a lanthanide tag for the analysis of protein dynamics by paramagnetic NMR spectroscopy.

Paramagnetic lanthanide tags potentially can enhance the effects of microsecond to millisecond dynamics in proteins on NMR signals and provide structural information on lowly populated states encoded in the pseudocontact shifts. We have investigated the microsecond to millisecond mobility of a two-point attached lanthanide tag, CLaNP-5, using paramagnetic (1)H CPMG relaxation dispersion methods. CLaNP-5 loaded with Lu(3+), Yb(3+), or Tm(3+) was attached to three sites on the surface of two proteins, pseudoazurin and cytochrome c. The paramagnetic center causes large relaxation dispersion effects for two attachment sites, suggesting that local dynamics of the protein at the attachment site causes mobility of the paramagnetic center. At one site the relaxation dispersions are small and limited to the immediate environment of the tag. It is concluded that paramagnetic relaxation dispersion could represent a sensitive method to probe protein dynamics. However, the selection of a rigid attachment site is of critical importance.

Modulation of protein-ligand interactions by photocleavage of a cyclic peptide using phosphatidylinositol 3-kinase SH3 domain as model system.

To photomodulate the interaction of the phosphatidylinositol 3-kinase SH3 domain with a peptide ligand, a cyclic peptide (cyclic-1) with a photolabile side chain-to-side chain linker was synthesized. The conformation of cyclic-1 differs from that of the parent linear peptide, but becomes identical by UV-irradiation. Accordingly, the binding affinity of cyclic-1 to the SH3 domain increased upon conversion of the cyclic to a linear flexible structure by irradiation (K(d): 3.4 +/- 1.7 and 0.9 +/- 0.3 mM, respectively). These results confirm the usefulness of a photocleavable peptide for photocontrol of peptide-protein interactions.

Design, synthesis, and evaluation of a lanthanide chelating protein probe: CLaNP-5 yields predictable paramagnetic effects independent of environment.

Immobilized lanthanide ions offer the opportunity to refine structures of proteins and the complexes they form by using restraints obtained from paramagnetic NMR experiments. We report the design, synthesis, and spectroscopic evaluation of the lanthanide chelator, Caged Lanthanide NMR Probe 5 (CLaNP-5) readily attachable to a protein surface via two cysteine residues. The probe causes tunable pseudocontact shifts, alignment, paramagnetic relaxation enhancement, and luminescence, by chelating it to the appropriate lanthanide ion. The observation of single shifts and the finding that the magnetic susceptibility tensors obtained from shifts and alignment analyses are highly similar strongly indicate that the probe is rigid with respect to the protein backbone. By placing the probe at various positions on a model protein it is demonstrated that the size and orientation of the magnetic susceptibility tensor of the probe are independent of the local protein environment. Consequently, the effects of the probe are readily predictable using a protein structure only. These findings designate CLaNP-5 as a protein probe to deliver unambiguous high quality structural restraints in studies on protein-protein and protein-ligand interactions.