Correlated inter-domain motions in adenylate kinase.

Correlated inter-domain motions in proteins can mediate fundamental biochemical processes such as signal transduction and allostery. Here we characterize at structural level the inter-domain coupling in a multidomain enzyme, Adenylate Kinase (AK), using computational methods that exploit the shape information encoded in residual dipolar couplings (RDCs) measured under steric alignment by nuclear magnetic resonance (NMR). We find experimental evidence for a multi-state equilibrium distribution along the opening/closing pathway of Adenylate Kinase, previously proposed from computational work, in which inter-domain interactions disfavour states where only the AMP binding domain is closed. In summary, we provide a robust experimental technique for study of allosteric regulation in AK and other enzymes.

RARRES3 suppresses breast cancer lung metastasis by regulating adhesion and differentiation.

In estrogen receptor-negative breast cancer patients, metastatic relapse usually occurs in the lung and is responsible for the fatal outcome of the disease. Thus, a better understanding of the biology of metastasis is needed. In particular, biomarkers to identify patients that are at risk of lung metastasis could open the avenue for new therapeutic opportunities. Here we characterize the biological activity of RARRES3, a new metastasis suppressor gene whose reduced expression in the primary breast tumors identifies a subgroup of patients more likely to develop lung metastasis. We show that RARRES3 downregulation engages metastasis-initiating capabilities by facilitating adhesion of the tumor cells to the lung parenchyma. In addition, impaired tumor cell differentiation due to the loss of RARRES3 phospholipase A1/A2 activity also contributes to lung metastasis. Our results establish RARRES3 downregulation as a potential biomarker to identify patients at high risk of lung metastasis who might benefit from a differentiation treatment in the adjuvant programme.

Average conformations determined from PRE data provide high-resolution maps of transient tertiary interactions in disordered proteins.

In the last decade it has become evident that disordered states of proteins play important physiological and pathological roles and that the transient tertiary interactions often present in these systems can play a role in their biological activity. The structural characterization of such states has so far largely relied on ensemble representations, which in principle account for both their local and global structural features. However, these approaches are inherently of low resolution due to the large number of degrees of freedom of conformational ensembles and to the sparse nature of the experimental data used to determine them. Here, we overcome these limitations by showing that tertiary interactions in disordered states can be mapped at high resolution by fitting paramagnetic relaxation enhancement data to a small number of conformations, which can be as low as one. This result opens up the possibility of determining the topology of cooperatively collapsed and hidden folded states when these are present in the vast conformational landscape accessible to disordered states of proteins. As a first application, we study the long-range tertiary interactions of acid-unfolded apomyoglobin from experimentally measured paramagnetic relaxation enhancement data.

Refinement of ensembles describing unstructured proteins using NMR residual dipolar couplings.

Residual dipolar couplings (RDCs) are unique probes of the structural and dynamical properties of biomolecules on the sub-millisecond time scale that can be used as restraints in ensemble molecular dynamics simulations to study the relationship between macromolecular motion and biological function. To date, however, this powerful strategy is applicable only to molecules that do not undergo shape changes on the time scale sampled by RDCs, thus preventing the study of key biological macromolecules such as multidomain and unstructured proteins. In this work, we circumvent this limitation by using an algorithm that explicitly computes the individual alignment tensors of the different ensemble members from their coordinates at each step in the simulation. As a first application, we determine an ensemble of conformations that accurately describes the structure and dynamics of chemically denatured ubiquitin. In analogy to dynamic refinement of folded, globular proteins, where simulations are initiated from average structures, we use statistical coil models as starting configuration because they represent the best available descriptions of unstructured proteins. We find that refinement with RDCs causes significant structural corrections and yields an ensemble that is in complete agreement with the measured RDCs and presents transient mid-range inter-residue interactions between strands beta1 and beta2 of the native protein, also observed in other studies based on trans-hydrogen bond (3)J(NC') scalar couplings and paramagnetic relaxation enhancements. Finally, and in spite of the high structural heterogeneity of the refined ensemble, we find that it can be cross-validated against RDCs not used to restrain the simulation. This method increases the range of systems that can be studied using ensemble simulations restrained by RDCs and is likely to yield new insights into how the large-scale motions of macromolecules relate to biological function.