Structural Biology of Cytoskeleton

Although all muscles use actin and myosin for contraction, only in skeletal and cardiac muscle are these proteins organized into sarcomeres, which are the fundamental contractile units of stri¬ated muscle. The Z-disc is the boundary of individual sarcomeres where thin filaments are anchored, and plays central and unique role as the main anchoring point of the molecular machinery that underlies muscle contraction. The Z-disc represents a highly organized three-dimensional structure containing several proteins assembled in multi-protein complexes, many of them centered on alpha-actinin. Alpha-actinin confers both stability and plasticity to a variety of actin-based arrays. In muscle cells it is a major component of the Z-disk, where it cross-links actin filaments and serves as a versatile platform for multiple protein-protein interactions. alpha-actinin binds the amino terminal end of the giant protein titin as well as to a plethora of Z-band proteins including myotilin, telethonin, MLP, FATZ and cypher/ZASP. Myotilin, MLP, FATZ, and ZASP bind alpha-actinin and telethonin binds titin and FATZ, which in turn binds also to gamma-filamin.

The principal experimental technique employed is macromolecular crystallography, in combination with biophysical and biochemical techniques. Complementary structural biology methods - NMR, FRET, electron microscopy, mass-spectrometry, circular dichroism, and small angle X-ray scattering, together with structural bioinformatics methods for structural analysis and validation are available in the Department, on the Campus or via collaborations and are recruited to enrich the structural information.

The goal of our research is to generate detailed structural information on the protein-protein interaction network in the stri¬ated muscle Z-disk, starting from its important component alpha-actinin and moving towards mac¬romolecular complexes to shed light on both functional and structural implications of these interactions.

Probing structure/function of enzymes via the pressure cell
It is well established that xenon and krypton can bind to many proteins at relatively modest partial pressures and can therefore be very useful as an alternative to heavy metals in an isomorphous replacement derivative search. For the purpose of pressurizing macromolecular crystals pressure cells have been developed that allow exposing crystal to pressures of several 10 atm. of a selected gas. The gas can be xenon or krypton, but a substrate, inhibitor of an enzyme rather than a modulator of pH in the crystal. We used a pressure cell to deliver CO2 to carbonic anhydrase crystals and observed the unprocessed substrate bound at the active site.

We plan to apply this method also on nitrous oxide reductase and on Cu,Zn SOD in order to probe the active site with gaseous substrates/inhibitors
 
Soft(er) X-rays in macromolecular crystallography
The fascinating possibility of solving structures by exploiting the enhancement of weak signals from several atoms naturally present in biological samples (calcium, iron, phosphorus and, in particular, sulfur), added to the more recent interest in elements like xenon, uranium and iodine, is leading researchers to consider the routine use of soft and softer X-rays in data-collection strategies. We aim at developing and/or implementing data-collection and data-analysis strategies to enhance the weak anomalous signal coming from atoms naturally present in biological samples. The in-house X-ray diffraction instrumentation (Bruker X8Proteum) as well as synchrotron radiation sources with beamlines offering soft(er) X-rays are being used for the purpose.
 
Protection from Reactive Oxygen Species
Cell damage is induced by reactive oxygen species (ROS). ROS are either free radicals, reactive anions containing oxygen atoms, or molecules containing oxygen atoms that can either produce free radicals or are chemically activated by them. Superoxide dismutases (SODs) are dedicated to keep the concentration of O2- in controlled low limits thus protecting biological molecules from oxidative damage. SODs are generally classified according to the metal species which acts as redox-active center to catalyze the dismutation reaction 2O2- + 2H+ ? O2 + H2O2. Superoxide dismutation can performed by three different families of superoxide dismutases which host distinct metal centers, and are unrelated in terms of amino-acid sequence and three-dimensional structure. Fe/Mn- SODs, which have ion or manganese in the active site, Cu,Zn SODs, with copper and zinc and the recently discovered NiSOD, which has nickel ion in the active site.

We have solved the structure of the Ni-containing SOD and are working on structural studies of Cu, Zn superoxide dismutases from pathogens, in particular M. tuberculosis and Haemophilus family in collaboration with the group of Andrea Battistoni, Department of Biology, University of Rome-2, Italy.

The future directions of this research are in the direction of SOD-like proteins which are distinguished by different levels of homology to the active cellular homologues. This structural information would allow to understand the molecular basis of the catalytically inactivity of these proteins.