The linkage between substrate orientation, dominated by remote interactions, and a sizable symmetrical region identified in all known ribosome structures indicates a guided rotatory moti
Trang 1P1—- FEBS Datta Plenary Lectureship Award
P1-001
Peptide bond formation, cotranslational
folding and antibiotics synergism
A Yonath
Structural Biology, Weizmann Inst., Rehovot, Israel
E-mail: ada.yonath@weizmann.ac.il
Ribosomes position their substrate at stereochemistry suitable for
peptide bond formation, and promote substrate-mediated cata-
lysis The linkage between substrate orientation, dominated by
remote interactions, and a sizable symmetrical region identified in
all known ribosome structures indicates a guided rotatory motion
of aminoacylated-tRNAs along a ribosomal path leadings to the
advance of nascent peptides into the protein exit tunnel at an
extended conformation The symmetry related region can transfer
intra-ribosomal signals between remote locations, since it con-
nects all ribosomal functionally sites These included the deco-
ding and peptide-bond-formation sites; the protein exit tunnel,
the tRNA entrance and exit environments and the protein exit
tunnel entrance The symmetry relates RNA backbone and nucle- otides orientation, but not sequence homology Thus, suggesting that ribosomes evolved by gene-fusion and demonstrates the superiority of the functional requirements over sequence conser- vation The protein exit tunnel acts as a dynamic functional entity capable of taking part in nascent protein elongation, dis- crimination, arrest and partial protein folding Initial steps in chaperon-aided cotranslational folding are associated with signifi- cant mobility of both the bacterial trigger factor and a ribosomal protein at the tunnel opening Similarly, major conformational alterations, induced by ribosomal recycling factor play a key role
in the termination steps of protein biosyntheses Comparative analysis of antibiotics binding modes to a eubacterial pathogen model and an archaeal sharing properties with eukaryotes showed that despite the overall conservation of the ribosome, phylogenetic and conformational variations in antibiotics binding pocket allow their selectivity, thus facilitating their therapeutical
usage
P2 - 50°" Anniversary IUBMB Lecture
P2-001
Protein misfolding and human disease: what
we have learned from 50 years of protein
science
C M Dobson
Department of Chemistry, University of Cambridge, Cambridge,
UK E-mail: cmd44(@cam.ac.uk
Proteins are the most abundant molecules in biology, other than
water, and enable or regulate all the chemical processes on
which life depends Over the past 50 years our knowledge and
understanding of these complex molecules has increased out of
all recognition The methods of X-ray diffraction, NMR spectr-
oscopy and electron microscopy, coupled with theoretical tech-
niques such as molecular dynamics simulations, have together
given us deep insight into their structures and properties In
addition, a very wide range of biophysical and biochemical
studies, particularly exploiting the power of protein engineering
techniques to probe the roles of individual amino acid side
chains, has revealed many of the intimate mechanistic details of
how individual protein molecules are able to exert their specific
functions In addition to the question of how the structures of
proteins are related to their functions, an additional question
emerged as soon as the first structures of proteins were solved
This question concerns the manner in which these fascinating
and intricate structures are attained by polypeptide chains fol-
lowing biosynthesis in the cell as an essentially unstructured
chain of amino acids This process of protein folding is of par-
ticular significance not just because it links gene sequences to
biological activity, but also because it represents perhaps the
most universal example of biomolecular self-assembly, a phe-
nomenon on which all life depends In recent years very consid-
erable progress has been made towards understanding the
fundamental basis of protein folding through the concerted application of a series of experimental approaches, notably the variety of biophysical techniques along with the methods of protein engineering, coupled with theoretical and computational approaches On the basis of these studies, the outline of a uni- versal and comprehensive mechanism of folding is emerging, and indeed is beginning to shed light on the way in which the amino acid sequence encodes the protein fold The development
of techniques to study protein folding has also resulted in major advances in our ability to define the structures and dynamics of proteins in states other than the native ones This topic is of considerable interest because these states are increasingly recog- nized as being coupled to many biological processes ranging from molecular trafficking to cell signalling and the regulation
of the cell cycle In addition, however, it has also become evi- dent that the failure to fold correctly, or to remain correctly folded, is the origin of a wide variety of human disorders ran- ging from Alzheimer’s disease to type II diabetes The study of such misfolding events and their consequences has been made possible by the adaptation of techniques developed to study the normal structural and folding characteristics of proteins As well
as shedding light on the nature of individual diseases, these studies have provided evidence for underlying generic aspects of protein misfolding and its consequences These conclusions are now providing insight into the origins of these diseases, why they are becoming epidemic in many parts of the world, and how they might be treated on a rational basis In addition they raise a series of fascinating issues about the underlying nature
of biological molecules and the driving forces of molecular evo- lution This lecture will attempt to bring these threads together
to give an overview of our present understanding of the nature
of protein molecules and how it has emerged over the past half
century
Trang 2References
1 Dobson CM Protein folding and misfolding Nature 2003;
426: 884-890
2 Dobson CM In the footsteps of alchemists Science 2004; 304:
1259-1262
3 Dobson CM Chemical space and biology Nature 2004; 432:
824-828
P3 —- Theodor Bucher Lecture and Medal
P3-001
Metabolomics, modelling and machine
learning in systems biology; understanding
complex systems using genetic programming
to produce simple interpretable rules The
Theodor Bucher Lecture and Medal
D B Kell
Chemistry, University of Manchester, Manchester, Lancs, UK
E-mail: dbk@manchester.ac.uk
Progress in Systems Biology — or in ‘understanding complex sys-
tems’ — depends on new technology [1-3], computational assist-
ance [4] and new philosophy [5], but probably not in that order
(pace [6]) Some developments include all three [7, 8]
References
1 Kell DB Metabolomics and systems biology: making sense of
the soup Curr Op Microbiol 2004; 7: 296-307
2 Goodacre R, Vaidyanathan S$, Dunn WB, Harrigan GG &
Kell DB Metabolomics by numbers: acquiring and under-
standing global metabolite data Trends Biotechnol 2004; 22:
245-252
P4 - PABMB Plenary Lecture
P4-001
Structure-based antibiotic design on the
bacterial membrane
N C J Strynadka
Biochemistry and Molecular Biology, University of British
Columbia, Vancouver, BC Canada
E-mail: natalie@byron.biochem.ubc.ca
Antibiotic resistance has become a major clinical problem
worldwide Our lab is interested in the structure-based design of
inhibitors which target antibiotic resistance mechanisms or novel
targets essential to bacterial pathogenesis The key determinant
of broad spectrum f-lactam resistance in Methicillin superbug
strains is the membrane spanning penicillin binding protein 2a
(PBP2a), a transpeptidase that is required to produce peptide
cross links that give the bacterial cell wall its necessary strength
and rigidity Due to its low affinity for B-lactams, PBP2a pro-
vides cross-linking transpeptidase activity at B-lactam concentra-
tions which inhibit the other cell-wall transpeptidases normally
produced by S aureus and other pathogenic bacteria The crys-
tal structures of native PBP2a from MRSA has been deter-
3 O'Hagan S, Dunn WB, Brown M, Knowles JD & Kell DB Closed-loop, multiobjective optimisation of analytical instru- mentation: gas-chromatography- time-of-flight mass spectro- metry of the metabolomes of human serum and of yeast fermentations Anal Chem 2005; 77: 290-303
4 Ihekwaba A, Broomhead DS, Grimley R, Benson N & Kell
DB Sensitivity analysis of parameters controlling oscillatory signalling in the NF-kappaB pathway: the roles of IKK and IkappaBalpha Systems Biology 2004; 1: 93-103
5 Kell DB & Oliver SG Here is the evidence, now what is the hypothesis? The complementary roles of inductive and hypoth- esis-driven science in the post-genomic era Bioessays 2004; 26:
99-105
6 Brenner 8 Nature June 5, 1980
7 King RD, Whelan KE, Jones FM, Reiser PGK, Bryant CH, Muggleton SH, Kell DB & Oliver SG Functional genomic hypothesis generation and experimentation by a robot scien- tist Nature 2004; 427: 247-252
8 Nelson DE, Ihekwaba A, Kell DB & White MRH Oscillations
in NF-kappaB signalling control the dynamics of target gene expression Science 2004; 306: 704-708
mined to 1.8 A resolution as well as acyl-enzyme complexes with various B-lactam antibiotic substrates An analysis of the PBP2a active site reveals the structural basis of its broad spec- trum resistance to the ~50 clinically utilized B-lactam antibiot- ics, and identifies features important for high affinity binding This information has been used in structure-based inhibitor design strategies aiming to combat MRSA resistance In terms
of novel targets, our laboratory has made significant progress
on the structural elucidation of the Type III secretion apparatus (TTSS) common to many Gram-negative pathogens The TTSS allows for the specific injection of bacterial proteins into human host cells, where they mediate their pathogenic effects Our laboratory has provided the first high resolution structures of TTSS proteins including the EspA translocation tube that spans from the bacteria OM to host membrane, the outer membrane secretin pilot protein and the inner membrane polymeric ring that is thought to act as the initial ‘platform’ upon which the other Type III components assemble These structures provide the foundation for understanding the molecular details of this fascinating pathogenic process as well as for the design of novel anti-microbials
Trang 3P5 - Sir Hans Krebs Lecture and Medal
P5-001
The epigenome in the context of the post-
genomic era
T Jenuwein
Research Institute of Molecular Pathology (IMP), The Vienna
Biocenter, Vienna, Austria E-mail: jenuwein@imp.univie.dac.at
The last years were highlighted by the landmark description of
the genomes of many model organisms, including the human gen-
ome These ‘genome projects’ have shown that more complex
eukaryotic organisms (e.g mammals) have a much bigger gen-
ome than less complex eukaryotes (e.g flies), although the
increased ‘biocomplexity’ is not reflected by an equivalent
increase in the number of protein coding genes Mechanisms
other than DNA sequence information have been adopted during
evolution to better index and regulate the various developmental
programmes and key regulatory processes, such as gene expres-
sion, chromosome segregation and cell division of eukaryotic
P6 -— Special Plenary Lecture
P6-001
Molecular mechanisms of bacterial swimming
and tumbling
K Namba
Protonic NanoMachine Group, Graduate School of Frontier
Biosciences, Osaka, Suita, Osaka Japan
E-mail: keiichi@fbs.osaka-u.ac jp
The bacterial flagellum is made of a rotary motor and a long
helical filament by means of which bacteria swim The flagellar
motor rotates at around 300 Hz and drives the rapid rotation of
each flagellum to propel the cell movements The long helical fil-
ament, which is a tubular structure with a diameter of about
20 nm, is made of a single protein flagellin The filament switches
between left- and right-handed helical forms in response to the
twisting force produced by reversal of the motor rotation, allow-
ing bacteria to alternate their swimming pattern between running
and tumbling for taxis The flagellum also has a short, highly
P7 —- EMBO Lecture
P7-001
Dynamics of spliceosome components in the
living cell nucleus
M Carmo-Fonseca
Institute of Molecular Medicine, University of Lisbon, Lisbon,
Portugal E-mail: carmo.fonseca@fm.ul pt
The spliceosome is a dynamic RNA-protein macromolecular
machine that is responsible for the splicing of intronic sequences
from pre-mRNA The spliceosome undergoes major structural
changes during the splicing reaction and its components must
be recycled for each new round of splicing Although the
spliceosome cycle has been extensively studied at the molecular
genomes In the nuclei of almost all eukaryotic cells, genomic DNA is highly folded and compacted with histone and non- histone proteins in a dynamic polymer called chromatin The discoveries that nucleosome remodelling machines and histone- modifying enzymes organize chromatin into accessible (euchro- matic) and inaccessible (heterochromatic) configurations reveal epigenetic mechanisms that considerably extend the information potential of the genetic code Thus, one genome can generate many — epigenomes — as the fertilized egg progresses through development and translates its information into a multitude of cell fates These epigenetic mechanisms are crucial for the func- tion of most, if not all, chromatin-templated processes and link alterations in the chromatin structure to gene regulation, X inac- tivation, chromosome organization and genome stability The implications of epigenetic research for human biology and dis- ease, including stem cells, cancer and aging are far-reaching and will form a modern foundation to explore the chromatin template
in a ‘post-genomic’ era
curved segment called hook, which connects the motor and the helical propeller Its bending flexibility makes it work as a nano- scale universal joint, while the filament is relatively more rigid to function as a propeller A very short segment made of proteins HAPI and HAP3 connects these two mechanically distinct struc- tures The flagellum is constructed by self-assembly of proteins translocated from the cytoplasm through the narrow central channel to the distal end of the growing structure, where one of three different cap complexes is attached to help efficient self- assembly of particular proteins that need to be assembled at each specific stage of the assembly process We have solved most part
of the structures in the cell exterior by X-ray crystallography, fiber diffraction and electron cryomicroscopy All these structures present interesting implications for the function of each molecule and subcomplex, demonstrating the importance of dual nature of protein molecules, dynamic flexibility and subatomic level preci-
sion
level, very little is known about the dynamics of spliceosome components in vivo We are using Fluorescence Recovery After Photobleaching (FRAP) to analyze the mobility and kinetic be- havior of spliceosome components in the nucleus of living human cells In addition we are performing Acceptor Photo- bleaching Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM) to visualize and spatially map the interactions between the splicing factors within the nucleoplasm (where splicing takes place) and in nuc- lear speckles (where splicing components accumulate when not engaged in splicing) Our results support the view that splicing factors assemble onto pre-spliceosome complexes localized in nuclear speckles
Trang 4P8 —- EMIBO Young Investigator Lecture
P8-001
Intracellular signaling in neutrophils and
osteoclasts
A Mocsai! and C A Lowell
"Department of Physiology, Semmelweis University, Budapest,
Hungary, *Department of Lab Medicine, University of California,
San Francisco, CA, USA E-mail: mocsai@ puskin.sote.hu
Immunoreceptors (BCR, TCR, Fe-receptors) signal by a common
mechanism whereby receptor-associated ITAM-bearing adaptors
become phosphorylated by Src-family kinases and then recruit
the Syk tyrosine kinase through its SH2-domains We found that
neutrophils lacking Src-family kinases, the ITAM-bearing adapt-
ers DAP12 and FeRy or the Syk tyrosine kinase failed to initiate
integrin-induced antimicrobial responses Phosphorylation of
DAP12 and FcRy was defective in cells lacking Sre-family kinas-
es, and the phosphorylation of Syk was absent both in Src-family
deficient and DAP127’ FcRy” cells We also found severe oste-
opetrosis in mice lacking both DAPI2 and FcRy DAPI2”~ FeRy’ double and Syk’ single mutant bone marrow cells failed to differentiate into mature osteoclasts and did not resorb bone DAP12 and FeRy were constitutively phosphorylated in wild type but not Sre-family deficient osteoclasts In turn, DAPI2 and FeRy were required for the constitutive phosphory- lation of Syk Retroviral expression of wild type Syk or DAP12 was able to restore osteoclast development and function in the relevant knockout background, but this functional reconstitution was abrogated by loss-of-function point mutations in the C-ter- minal SH2 domain of Syk or in the two tyrosines within the DAP12 ITAM motif These results suggest that integrin-mediated antimicrobial responses of neutrophils and the development and function of osteoclasts require an immunoreceptor-like signaling mechanism, whereby Src-family mediated phosphorylation of DAPI2 and FeRy leads to a phospho-ITAM dependent activa- tion of Syk, which is in turn required for downstream signaling and functional responses