Macromolecular session at ECM24
The scientific program of ECM24 featured many exciting new macromolecular structure determinations. As the barriers to preparation of single crystals of integral membrane proteins continue to fall details concerning processes and reactions that take place within membranes are coming to light. Satohi Murakami (Japan) presented the structure of a multidrug efflux transporter (AcrB) that is responsible for antibiotic resistance. This protein, a multidrug transporter in E. coli, cooperates with an outer-membrane channel, TolC, and a membrane-fusion protein, AcrA, to form a supra-molecular complex that penetrates the cytoplasmic membrane and the outer membrane. In the first crystal structure of AcrB, solved in 2002, the active homo-trimer had threefold crystallographic symmetry. Structural analysis on a new crystal form having a trimer in the asymmetric unit revealed that each of the monomers have different conformations corresponding to one of the three functional states of the transport cycle. This asymmetric property is very important to the molecular mechanism of transport. Drugs are exported by a three-step rotating mechanism in which substrates undergo changes in ordered binding. In many cases, the activity of a symmetric multimeric protein requires a loss of symmetry. Allostericity and cooperativity are common examples and smart strategies to control protein function through a balance of symmetric and asymmetric forms.
Sin Urban (Johns Hopkins U., USA) reported on his structure determination of a serine protease from the Rhomboid family of seven stranded transmembrane proteins. Rhomboid proteins are membrane-embedded enzymes with the unusual ability to cleave transmembrane protein segments within the membrane. This form of intramembrane proteolysis plays key roles in diverse cell communication events including EGF signaling during animal development, and quorum sensing during bacterial growth. The structure seems to be preserved throughout evolution. The site of serine cleavage is 10Å inside the membrane in a water filled cavity. The existence of two different crystal forms suggests mechanisms of action that can be tested.
In a session on structure validation and quality control, Alex Wlodawer (NCI, USA) discussed the perils of using molecular restraints in protein structure refinement that can lead to errors in structural details related to function. E. Chabriere (CNRS, France) described the serendipitous determination of the structure of a member of a protein family that has the controversial and puzzling characteristic of being ubiquitous in eukaryotes but absent from eukaryotic genomes. The presentation also illustrated the power of crystallography to sequence proteins of unknown sequence with the help of mass spectroscopy.
Other remarkable presentations in the macromolecular microsymposia included an overview of the complex conjugation process in the ubiquitin protein family (T. Sixma, Netherlands), details of the initial stages of ribosomal function (L. Jenner, U. of Strasbourg, France), and high throughput ligand screening to simultaneously improve crystallization and identify substrate preference (A. Allali Hassani, Canada). Reports of systematic analysis of large ensembles of structural data included Gideon Schreiber's (Weizmann Inst., Israel) analysis of specific interactions at protein/protein interfaces used to generate contact maps and identify recurring graph motifs, powerful new protein alignment techniques being developed by J. Söding (Germany) and Christine Orengo's (U. College, UK) use of structural genomics to explore the evolution of protein function. One of the most complex structures described at ECM24 was that of frataxin, a protein important to iron delivery and detoxification in which twenty four copies of the protein form the iron binding complex. On the basis of a single crystal study of a trimer of iron free and iron complexed fratoxin and a single particle electron microscopy reconstruction of the 24 subunit form, S. Al Karagaghi (Lund U., Sweden) discussed the mechanics of fratoxin self assembly, iron acquition, detoxification and storage and how mutation in patients with Freedrichs atoxia may destabalize the active trimer.William L. Duax