Ernest D. Laue

The CCPN data model for NMR spectroscopy: Development of a software pipeline

To address data management and data exchange problems in the nuclear magnetic resonance (NMR) community, the Collaborative Computing Project for the NMR community (CCPN) created a “Data Model” that describes all the different types of information needed in an NMR structural study, from molecular structure and NMR parameters to coordinates. This paper describes the development of a set of software applications that use the Data Model and its associated libraries, thus validating the approach. These applications are freely available and provide a pipeline for high‐throughput analysis of NMR data. Three programs work directly with the Data Model: CcpNmr Analysis, an entirely new analysis and interactive display program, the CcpNmr FormatConverter, which allows transfer of data from programs commonly used in NMR to and from the Data Model, and the CLOUDS software for automated structure calculation and assignment (Carnegie Mellon University), which was rewritten to interact directly with the Data Model. The ARIA 2.0 software for structure calculation (Institut Pasteur) and the QUEEN program for validation of restraints (University of Nijmegen) were extended to provide conversion of their data to the Data Model. During these developments the Data Model has been thoroughly tested and used, demonstrating that applications can successfully exchange data via the Data Model. The software architecture developed by CCPN is now ready for new developments, such as integration with additional software applications and extensions of the Data Model into other areas of research. Proteins 2005.

Single-cell Hi-C reveals cell-to-cell variability in chromosome structure

Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture (3C) assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single-cell Hi-C, combined with genome-wide statistical analysis and structural modelling of single-copy X chromosomes, to show that individual chromosomes maintain domain organization at the megabase scale, but show variable cell-to-cell chromosome structures at larger scales. Despite this structural stochasticity, localization of active gene domains to boundaries of chromosome territories is a hallmark of chromosomal conformation. Single-cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organization underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns.

Structure of the HP1 chromodomain bound to histone H3 methylated at lysine 9

Specific modifications to histones are essential epigenetic markers—heritable changes in gene expression that do not affect the DNA sequence. Methylation of lysine 9 in histone H3 is recognized by heterochromatin protein 1 (HP1), which directs the binding of other proteins to control chromatin structure and gene expression. Here we show that HP1 uses an induced-fit mechanism for recognition of this modification, as revealed by the structure of its chromodomain bound to a histone H3 peptide dimethylated at Nζ of lysine 9. The binding pocket for the N-methyl groups is provided by three aromatic side chains, Tyr 21, Trp 42 and Phe 45, which reside in two regions that become ordered on binding of the peptide. The side chain of Lys 9 is almost fully extended and surrounded by residues that are conserved in many other chromodomains. The QTAR peptide sequence preceding Lys 9 makes most of the additional interactions with the chromodomain, with HP1 residues Val 23, Leu 40, Trp 42, Leu 58 and Cys 60 appearing to be a major determinant of specificity by binding the key buried Ala 7. These findings predict which other chromodomains will bind methylated proteins and suggest a motif that they recognize.

Heterochromatin Dynamics in Mouse Cells: Interaction between Chromatin Assembly Factor 1 and HP1 Proteins

Mechanisms contributing to the maintenance of heterochromatin in proliferating cells are poorly understood. We demonstrate that chromatin assembly factor 1 (CAF-1) binds to mouse HP1 proteins via an N-terminal domain of its p150 subunit, a domain dispensable for nucleosome assembly during DNA replication. Mutations in p150 prevent association with HP1 in heterochromatin in cells that are not in S phase and the formation of CAF-1-HP1 complexes in nascent chromatin during DNA replication in vitro. We suggest that CAF-1 p150 has a heterochromatin-specific function distinct from its nucleosome assembly function during S phase. Just before mitosis, CAF-1 p150 and some HP1 progressively dissociate from heterochromatin concomitant with histone H3 phosphorylation. The HP1 proteins reassociate with chromatin at the end of mitosis, as histone H3 is dephosphorylated.

The structure of mouse HP1 suggests a unique mode of single peptide recognition by the shadow chromo domain dimer

The heterochromatin protein 1 (HP1) family of proteins is involved in gene silencing via the formation of heterochromatic structures. They are composed of two related domains: an N‐terminal chromo domain and a C‐terminal shadow chromo domain. Present results suggest that chromo domains may function as protein interaction motifs, bringing together different proteins in multi‐protein complexes and locating them in heterochromatin. We have previously determined the structure of the chromo domain from the mouse HP1β protein, MOD1. We show here that, in contrast to the chromo domain, the shadow chromo domain is a homodimer. The intact HP1β protein is also dimeric, where the interaction is mediated by the shadow chromo domain, with the chromo domains moving independently of each other at the end of flexible linkers. Mapping studies, with fragments of the CAF1 and TIF1β proteins, show that an intact, dimeric, shadow chromo domain structure is required for complex formation.

Crystal structure of a small G protein in complex with the GTPase-activating protein rhoGAP

Small G proteins transduce signals from plasma-membrane receptors to control a wide range of cellular functions,. These proteins are clustered into distinct families but all act as molecular switches, active in their GTP-bound form but inactive when GDP-bound. The Rho family of G proteins, which includes Cdc42Hs, activate effectors involved in the regulation of cytoskeleton formation, cell proliferation and the JNK signalling pathway. G proteins generally have a low intrinsic GTPase hydrolytic activity but there are family-specific groups of GTPase-activating proteins (GAPs) that enhance the rate of GTP hydrolysis by up to 105times,. We report here the crystal structure of Cdc42Hs, with the non-hydrolysable GTP analogue GMPPNP, in complex with the GAP domain of p50rhoGAP at 2.7 å resolution. In the complex Cdc42Hs interacts, mainly through its switch I and II regions, with a shallow pocket on rhoGAP which is lined with conserved residues. Arg 85 of rhoGAP interacts with the P-loop of Cdc42Hs, but from biochemical data and by analogy with the G-protein subunit Giα1 (ref. 12), we propose that it adopts a different conformation during the catalytic cycle which enables it to stabilize the transition state of the GTP-hydrolysis reaction.

Structural basis of HP1/PXVXL motif peptide interactions and HP1 localisation to heterochromatin

HP1 family proteins are adaptor molecules, containing two related chromo domains that are required for chromatin packaging and gene silencing. Here we present the structure of the chromo shadow domain from mouse HP1β bound to a peptide containing a consensus PXVXL motif found in many HP1 binding partners. The shadow domain exhibits a novel mode of peptide recognition, where the peptide binds across the dimer interface, sandwiched in a β‐sheet between strands from each monomer. The structure allows us to predict which other shadow domains bind similar PXVXL motif‐containing peptides and provides a framework for predicting the sequence specificity of the others. We show that targeting of HP1β to heterochromatin requires shadow domain interactions with PXVXL‐containing proteins in addition to chromo domain recognition of Lys‐9‐methylated histone H3. Interestingly, it also appears to require the simultaneous recognition of two Lys‐9‐methylated histone H3 molecules. This finding implies a further complexity to the histone code for regulation of chromatin structure and suggests how binding of HP1 family proteins may lead to its condensation.

Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell-cycle inhibitor p19INK4d.

The crystal structure of the cyclin D-dependent kinase Cdk6 bound to the p19INK4d protein has been determined at 1.9 Å resolution. The results provide the first structural information for a cyclin D-dependent protein kinase and show how the INK4 family of CDK inhibitors bind. The structure indicates that the conformational changes induced by p19INK4d inhibit both productive binding of ATP and the cyclin-induced rearrangement of the kinase from an inactive to an active conformation. The structure also shows how binding of an INK4 inhibitor would prevent binding of p27Kip1, resulting in its redistribution to other CDKs. Identification of the critical residues involved in the interaction explains how mutations in Cdk4 and p16INK4a result in loss of kinase inhibition and cancer.

Exponential sampling, an alternative method for sampling in two-dimensional NMR experiments

Abstract A new method for sampling in two-dimensional nuclear magnetic resonance experiments is proposed and tested using one-dimensional spectra as models. The free induction decays are sampled exponentially, using many points where the signal-to-noise ratio (S/N) is high and a few where it is low. Using the maximum entropy method to reconstruct spectra, much higher resolution can be obtained than by using conventional sampling (for a given number of data points). The method is shown to work for FIDs having even very poor S/N. It should prove valuable in the future for 2D NMR experiments where at present valuable high-resolution information is lost as a result of the necessity for truncation of data sets in t1 in order to optimize sensitivity.

Structure of Cdc42 bound to the GTPase binding domain of PAK

The Rho family GTPases, Cdc42, Rac and Rho, regulate signal transduction pathways via interactions with downstream effector proteins. We report here the solution structure of Cdc42 bound to the GTPase binding domain of αPAK, an effector of both Cdc42 and Rac. The structure is compared with those of Cdc42 bound to similar fragments of ACK and WASP, two effector proteins that bind only to Cdc42. The N-termini of all three effector fragments bind in an extended conformation to strand β2 of Cdc42, and contact helices α1 and α5. The remaining residues bind to switches I and II of Cdc42, but in a significantly different manner. The structure, together with mutagenesis data, suggests reasons for the specificity of these interactions and provides insight into the mechanism of PAK activation.