STRUCTURE AND GENOME RELEASE MECHANISM OF HUMAN CARDIOVIRUS SAFFOLD VIRUS-3 (SAFV-3)

Saffold vi rus (SAFV) is the hu man Cardiovirus closely re lated to the Theiler murine encephalomyelitis vi rus (TMEV), of the fam ily picornaviridae (1). It was re ported that, SAFV might cause re spi ra tory, gas tro in tes ti nal, and cen tral ner vous sys tem in fec tions (1,2). To date 11 ge no types of SAFV have been iden ti fied (1, 3). In the pres ent study, the three-di men sional struc ture of SAFV-3 has been de ter mined at 2.5 C res o lu tion. Al though the ar chi tec ture of the ma jor capsid pro teins VP1, VP2 and VP3 of SAFV-3 is sim i lar to other cardioviruses, there are some dif fer ences on the sur face loops. The pres ence of disulphide bond on the sur face of VP3, sur pris ingly di min ish the sta bil ity and infectivity of SAFV-3. Sev eral capsid-bind ing and rep li ca tion in hib i tors of other picornaviruses fail to have any ef fect on SAFV-3. It was also shown that SAFV-3 dis so ci ates in to pentameric sub units upon the ge nome re lease.

Nat u ral killer cells (NK cells) are large gran u lar lym phocytes -a type of white blood cells. They are able to kill virally in fected, stressed or tu mor cells. Un like T-cells, the ac tiv ity of NK cells is in nate, they do not need to have previ ous ex pe ri ence with a tu mor -they are nat u ral kill ers. NKR-P1 (CD161) is a re cep tor on a sur face of hu man NK cells. LLT1 is a ligand for NKR-P1 re cep tor, ex pressed pri mar ily on ac ti vated lym pho cytes and an ti gen pre sent ing cells. The in ter ac tion of the ligand with the re cep tor in hibits NK cell cytotoxicity; how ever, it may have also ac ti vation ef fects in some cases. Extracellular do mains of both bind ing part ners, NKR-P1 and LLT1, have C-type lectin like (CTL) fold.
Us ing X-ray dif frac tion, we de ter mined four struc tures of LLT1 [1] from pro tein pro duced in HEK293S GnTI-cells. The pro tein with GlcNAc 2 Man 5 glycosylation packs into hexamers (con sist ing of three dimers) in crys tals. The pro tein deglycosylated af ter the first N-acetylglucosamine was found in our crys tal struc tures in forms of dimers (in pH 7.0) and mono mers (in pH 3.5).
The LLT1 struc tures (Fig ure 1) show that LLT1 follows the "clas si cal" mode of dimerization known from other struc tures with the same fold (CD69 [2], Clr-g [3]). The se ries of the LLT1 struc tures bring in sight into variabil ity of the dimerization in ter face, flex i bil ity of the outer long loop of the CTL do main and in flu ence of glycosylation on the structure.
This study was sup ported by BIOCEV CZ. 1 Po tas sium ion (K + ) up take in yeast is me di ated mainly by the Trk1/2 pro teins that en able cells to sur vive on ex ter nal K + con cen tra tion as low as a few µM. Fun gal Trks are related to prokaryotic TRK and Ktr and plant HKT K + transport sys tems (the SKT pro tein fam ily). Over all se quence sim i lar ity is very low, thus re quir ing ex per i men tal ver i fi cation of homology mod els. Here a re fined struc tural model of the Saccharomyces cerevisiae Trk1 is pre sented that was ob tained by com bin ing homology mod el ing, mo lec u lar dynam ics sim u la tion and ex per i men tal ver i fi ca tion through func tional anal y sis of mu tants. Struc tural mod els and exper i men tal re sults showed that glycines within the se lec tivity fil ter, con served amongst the K-chan nel/trans porter fam ily, are not only im por tant for pro tein func tion, but are also re quired for cor rect fold ing/ mem brane tar get ing. A con served as par tic acid in the P A he lix (D79) and a lysine in the M2 D he lix (K1147) were pro posed ear lier to in ter act. Our re sults sug gested in di vid ual roles of these resi dues in fold ing, struc tural in teg rity and func tion. While mu ta tions of D79 com pletely abol ished pro tein fold ing, mu ta tions at po si tion 1147 were tol er ated to some ex tent. In trigu ingly, a sec ond ary in ter ac tion of D79 with R76 could en hance fold ing/sta bil ity of Trk1 and enable a fraction of Trk1[K1147A] to fold.
The part of the ion per me ation path con tain ing the selec tiv ity fil ter is shaped sim i lar to that of ion chan nels. How ever be low the se lec tiv ity fil ter it is ob structed or regu lated by a proline con tain ing loop. The pre sented model could pro vide the struc tural ba sis for ad dress ing the long stand ing ques tion if Trk1 is a pas sive or ac tive ion-translocation system.

Uppsala Uni ver sity and The Eu ro pean XFEL
At the be gin ning of 2017, the Eu ro pean X-ray Free-Electron-La ser (XFEL) in Ham burg will be gin user op er a tions. Free-elec tron la sers are the most bril liant sources of X-rays to date, ex ceed ing the peak bril liance of con ven tional synchrotrons by a fac tor of 10 bil lion, and im prov ing. In the du ra tion of a sin gle flash, the beam fo cused to a micron-sized spot has the same power den sity as all the sunlight hit ting the Earth, fo cused to a milli metre square. The in ter ac tion of an in tense X-ray pulse with mat ter is profoundly dif fer ent from that of an op ti cal pulse. A nec es sary goal of re search with these ma chines is to ex plore photon-ma te rial in ter ac tions in strong X-ray fields. The aim in bi ol ogy is to step be yond con ven tional dam age lim its and de velop the sci ence and tech nol ogy re quired to en able high-res o lu tion im ag ing of both crys tal line and non-crystal line bi o log i cal ob jects at high res o lu tion. El i gi ble tar gets in clude sin gle vi rus par ti cles, organelles, cells, nanocrystals, en gi neered nanoclusters and iso lated macromolecules. The talk will sum ma rise de vel op ments at the Eu ro pean XFEL and pro vide an over view of some of the bi o log i cal re sults from the Linac Co her ent Light Source (LCLS), the first hard X-ray free-elec tron la ser. One of the aims of the talk us ex plore pos si bil i ties for in ter ested Czech sci en tists to par tic i pate in rev o lu tion ary new ex per i ments at the Eu ro pean XFEL. Struc tural bi ol ogy of to day is a well-de fined field of science. It is not so for bioinformatics, which is un der stood from very nar row clas si cal view (in for ma tics of the genome) on the one hand, to very wide con cept of in for ma tics of any bi ol ogy re lated in for ma tion. In all cases, bio informatics be comes an im por tant field of sci ence as the amount of bio-re lated in for ma tion, es pe cially from Next Gen er ation Se quenc ing (NGS), is in creas ing dra mat i cally, and, for the time be ing, there is no soft ware tool avail able that would be able to ex tract all the bi o log i cal in for ma tion hidden in the data. In con trary, struc tural bioinformatics is rel a tively well de fined part of bioinformatics (see, for ex am ple [1][2]), which is re lated to the anal y sis and pre dic tion of the threedi men sional struc ture of bi o log i cal macromolecules.The term struc tural has the same mean ing as in struc tural bi ology, and struc tural bioinformatics can be seen as a part of com pu ta tional struc tural bi ol ogy. Even if the grow of 3D struc tural data is much lower com pared to NGS, also here the in crease is ex po nen tial and calls for new ap proaches to ex tract struc tur ally and/or biologically relevant in for mation.
In our group, we have de vel oped sev eral soft ware tools that are able to help in solv ing such a task. These are Motive Query [3] for quick find ing and ex trac tion of biomacromolecular frag ments, SiteBinder [4] for fast and ac cu rate com par i son of these frag ments, MotiveValidator [5] and ValidatorDB [6] for val i da tion of lig ands and nonstan dard res i dues, and AtomicChargeCalculator [7] for cal cu la tion of par tial atomic charges. Last but not least, we have de vel oped also MOLE [8], a soft ware tool for de tection and char ac ter iza tion of chan nels and pores in biomacromolecules. All the soft ware tools are ac ces si ble from the link http://ncbr.muni.cz/WebChemistry.