Jose M. Martin-Garcia

Molecular Basis of Histone H3K4me3 Recognition by ING4

The inhibitors of growth (ING) family of tumor suppressors consists of five homologous proteins involved in chromatin remodeling. They form part of different acetylation and deacetylation complexes and are thought to direct them to specific regions of the chromatin, through the recognition of H3K4me3 (trimethylated K4 in the histone 3 tail) by their conserved plant homeodomain (PHD). We have determined the crystal structure of ING4-PHD bound to H3K4me3, which reveals a tight complex stabilized by numerous interactions. NMR shows that there is a reduction in the backbone mobility on the regions of the PHD that participate in the peptide binding, and binding affinities differ depending on histone tail lengths Thermodynamic analysis reveals that the discrimination in favor of methylated lysine is entropy-driven, contrary to what has been described for chromodomains. The molecular basis of H3K4me3 recognition by ING4 differs from that of ING2, which is consistent with their different affinities for methylated histone tails. These differences suggest a distinct role in transcriptional regulation for these two ING family members because of the antagonistic effect of the complexes that they recruit onto chromatin. Our results illustrate the versatility of PHD fingers as readers of the histone code.

Symmetric hyperbolicity and consistent boundary conditions for second-order Einstein equations

We present two families of first-order in time and second-order in space formulations of the Einstein equations (variants of the Arnowitt-Deser-Misner formulation) that admit a complete set of characteristic variables and a conserved energy that can be expressed in terms of the characteristic variables. The associated constraint system is also symmetric hyperbolic in this sense, and all characteristic speeds are physical. We propose a family of constraint-preserving boundary conditions that is applicable if the boundary is smooth with tangential shift. We conjecture that the resulting initial-boundary value problem is well-posed.

Gauge-invariant and coordinate-independent perturbations of stellar collapse: The interior

Small nonspherical perturbations of a spherically symmetric but time-dependent background spacetime can be used to model situations of astrophysical interest, for example the production of gravitational waves in a supernova explosion. We allow for perfect fluid matter with an arbitrary equation of state p=p(?,s), coupled to general relativity. Applying a general framework proposed by Gerlach and Sengupta, we obtain covariant field equations, in a 2+2 reduction of the spacetime, for the background and a complete set of gauge-invariant perturbations, and then scalarize them using the natural frame provided by the fluid. Building on previous work by Seidel, we identify a set of true perturbation degrees of freedom admitting free initial data for the axial and for the l>~2 polar perturbations. The true degrees of freedom are evolved among themselves by a set of coupled wave and transport equations, while the remaining degrees of freedom can be obtained by quadratures. The polar l=0,1 perturbations are discussed in the same framework. They require gauge fixing and do not admit an unconstrained evolution scheme.

Symmetric hyperbolic form of systems of second-order evolution equations subject to constraints

Motivated by the initial-boundary value problem for the Einstein equations, we propose a definition of symmetric hyperbolicity for systems of evolution equations that are first order in time but second order in space. This can be used to impose constraint-preserving boundary conditions. The general methods are illustrated in detail in the toy model of electromagnetism.

Serial femtosecond crystallography: A revolution in structural biology ☆

Macromolecular crystallography at synchrotron sources has proven to be the most influential method within structural biology, producing thousands of structures since its inception. While its utility has been instrumental in progressing our knowledge of structures of molecules, it suffers from limitations such as the need for large, well-diffracting crystals, and radiation damage that can hamper native structural determination. The recent advent of X-ray free electron lasers (XFELs) and their implementation in the emerging field of serial femtosecond crystallography (SFX) has given rise to a remarkable expansion upon existing crystallographic constraints, allowing structural biologists access to previously restricted scientific territory. SFX relies on exceptionally brilliant, micro-focused X-ray pulses, which are femtoseconds in duration, to probe nano/micrometer sized crystals in a serial fashion. This results in data sets comprised of individual snapshots, each capturing Bragg diffraction of single crystals in random orientations prior to their subsequent destruction. Thus structural elucidation while avoiding radiation damage, even at room temperature, can now be achieved. This emerging field has cultivated new methods for nanocrystallogenesis, sample delivery, and data processing. Opportunities and challenges within SFX are reviewed herein.

All nonspherical perturbations of the Choptuik spacetime decay

We study the nonspherical linear perturbations of the discretely self-similar and spherically symmetric solution for a self-gravitating scalar field discovered by Choptuik in the context of marginal gravitational collapse. We find that all nonspherical perturbations decay. Therefore critical phenomena at the threshold of gravitational collapse, originally found in spherical symmetry, will extend to (at least slightly) nonspherical initial data.

Crystallographic structure of the SH3 domain of the human c‐Yes tyrosine kinase: Loop flexibility and amyloid aggregation

SH3 domains from the Src family of tyrosine kinases represent an interesting example of the delicate balance between promiscuity and specificity characteristic of proline‐rich ligand recognition by SH3 domains. The development of inhibitors of therapeutic potential requires a good understanding of the molecular determinants of binding affinity and specificity and relies on the availability of high quality structural information. Here, we present the first high‐resolution crystal structure of the SH3 domain of the c‐Yes oncogen. Comparison with other SH3 domains from the Src family revealed significant deviations in the loop regions. In particular, the n‐Src loop, highly flexible and partially disordered, is stabilized in an unusual conformation by the establishment of several intramolecular hydrogen bonds. Additionally, we present here the first report of amyloid aggregation by an SH3 domain from the Src family.

Global structure of Choptuik's critical solution in scalar field collapse

At the threshold of black hole formation in the gravitational collapse of a scalar field a naked singularity is formed through a universal critical solution that is discretely self-similar. We study the global spacetime structure of this solution. It is spherically symmetric, discretely self-similar, regular at the center to the past of the singularity, and regular at the past light cone of the singularity. At the future light cone of the singularity, which is also a Cauchy horizon, the curvature is finite and continuous but not differentiable. To the future of the Cauchy horizon the solution is not unique, but depends on a free function (the null data coming out of the naked singularity). There is a unique continuation with a regular center (which is self-similar). All other self-similar continuations have a central timelike singularity with negative mass.

Structural enzymology using X-ray free electron lasers

Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.

Proliferating cell nuclear antigen (PCNA) interactions in solution studied by NMR.

PCNA is an essential factor for DNA replication and repair. It forms a ring shaped structure of 86 kDa by the symmetric association of three identical protomers. The ring encircles the DNA and acts as a docking platform for other proteins, most of them containing the PCNA Interaction Protein sequence (PIP-box). We have used NMR to characterize the interactions of PCNA with several other proteins and fragments in solution. The binding of the PIP-box peptide of the cell cycle inhibitor p21 to PCNA is consistent with the crystal structure of the complex. A shorter p21 peptide binds with reduced affinity but retains most of the molecular recognition determinants. However the binding of the corresponding peptide of the tumor suppressor ING1 is extremely weak, indicating that slight deviations from the consensus PIP-box sequence dramatically reduce the affinity for PCNA, in contrast with a proposed less stringent PIP-box sequence requirement. We could not detect any binding between PCNA and the MCL-1 or the CDK2 protein, reported to interact with PCNA in biochemical assays. This suggests that they do not bind directly to PCNA, or they do but very weakly, with additional unidentified factors stabilizing the interactions in the cell. Backbone dynamics measurements show three PCNA regions with high relative flexibility, including the interdomain connector loop (IDCL) and the C-terminus, both of them involved in the interaction with the PIP-box. Our work provides the basis for high resolution studies of direct ligand binding to PCNA in solution.