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The L55P transthyretin (TTR) familial amyloid polyneuropathy-associated variant is distinct from the other TTR variants studied to date and the wild-type protein in that the L55P tetramer can dissociate to the monomeric amyloidogenic intermediate and form fibril precursors under physiological conditions (pH 7.0, 37°C). The activation barrier associated with L55P-TTR tetramer dissociation is lower than the barrier for wild-type transthyretin dissociation, which does not form fibrils under physiological conditions. The L55P-TTR tetramer is also very sensitive to acidic conditions, readily dissociating to form the monomeric amyloidogenic intermediate between pH 5.5-5.0 where the wildtype TTR adopts a nonamyloidogenic tetrameric structure. The formation of the L55P monomeric amyloidogenic intermediate involves subtle tertiary structural changes within the -sheet rich subunit as discerned from Trp fluorescence, circular dichroism analysis, and ANS binding studies. The assembly of the L55P-TTR amyloidogenic intermediate at physiological pH (pH 7.5) affords protofilaments that elongate with time. TEM studies suggest that the entropic barrier associated with filament assembly (amyloid fibril formation) is high in vitro, amyloid being defined by the laterally assembled four filament structure observed by Blake upon isolation of "fibrils" from the eye of a FAP patient. The L55P-TTR protofilaments formed in vitro bind Congo red and thioflavin T (albeit more weakly than the fibrils produced at acidic pH), suggesting that the structure observed probably represents an amyloid precursor. The structural continuum from misfolded monomer through protofilaments, filaments, and ultimately fibrils must be considered as a possible source of pathology associated with these diseases.There are several human neurodegenerative diseases putatively caused by extracellular protein deposition (1). The most familiar of these include the amyloid diseases, e.g. Alzheimer's disease and senile systemic amyloidosis (2-7). In all amyloid diseases, proteins undergo conformational changes either before or coincident with their self-assembly into highly ordered amyloid fibrils. Amyloid fibrils having a cross -sheet quaternary structure are typically deposited in the brain and/or in peripheral tissues and have been linked to neurodegeneration and/or organ dysfunction by genetic evidence (2,(8)(9)(10)(11). The amyloid hypothesis implicates amyloid fibrils as the causative agent in amyloid diseases through a gain of function phenotype. (1,7,9,(12)(13)(14). Recent results from several laboratories, including our own, demonstrate the existence of preamyloid quaternary structural intermediates (protofilaments), implying that the whole process of amyloid fibril formation must be considered as a possible cause of pathology, including the first step in the process, which is protein misfolding in the case of structurally well-defined amyloid proteins (14-21).Understanding the mechanism of how a folded protein is converted into an insoluble amyloid fibr...

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Herpes simplex virus type 1 (HSV 1) 1 thymidine kinase (TK) is a multifunctional enzyme that possesses kinase activities normally performed by three separate cellular enzymes. It phosphorylates thymidine (dT), which is then transformed by cellular kinases to the triphosphorylated DNA building block, and deoxyuridine (dU); both reactions are comparable to the function of human cellular TK. Further, it converts deoxycytidine (dC) to dCMP, as does human deoxycytidine kinase (dCK), and phosphorylates thymidylate (dTMP), as does human TMP kinase (TmpK) (1-3). Moreover, unlike its cellular counterpart human cellular TK, HSV 1 TK is able to phosphorylate pyrimidine, as well as purine analogs, and discloses low stereochemical demands for the ribose moiety, as it also accepts acyclic side chains as phosphoryl group acceptors e.g. (4 -6). These differences in substrate diversity are the crucial molecular basis for the selective treatment of viral infections. Nowadays, the most widely used therapeutic compounds to interfere with a severe HSV 1 infection are the purine analogs acyclovir (ACV) and penciclovir and their prodrugs valaciclovir and famciclovir, respectively. They require HSV 1 TK to be efficiently activated in order to block virus proliferation by inhibition of viral DNA polymerase. HSV 1 TK is the key enzyme in this antiviral strategy. In gene therapy of cancer (7,8) and AIDS (9), HSV 1 TK is used as a suicide enzyme in combination with the purine analog ganciclovir. Another important application is the use of HSV 1 TK as a rescue system in allogeneic bone marrow transplantation-induced graft versus host disease (10). In addition to the significance from a therapeutic point of view, HSV 1 TK seems to be important for the reactivation of the virus from lifelong latent infection in neuronal ganglia (11-13). However, there is evidence that human TK can functionally replace viral TK in terms of reactivation of the virus from latency (14).There are no recognizable sequence similarities between HSV 1 TK and human cellular TK (15). Rather, sequence alignments have detected similarities between herpesvirus TKs and human dCK (16) and to a lesser extent cellular TmpK (17). Despite the limited sequence homology with enzymes of the nucleotide kinase (NK) family, HSV 1 TK shares structural features comprising a parallel five-stranded ␤-sheet and a glycine-rich loop common to all NKs. In the crystal structure, HSV 1 TK is a homodimeric enzyme with 376 amino acids per subunit (18 -20). The two subunits are related by C2 symmetry. The active site is formed by an ATP-and a nucleoside-binding region. The visual representation of the thymidine binding site is depicted in Fig. 1, featuring a complex hydrogen bond network within the active site. The thymine ring makes pairwise hydrogen bond interaction via its 4-carbonyl and 3-NH group with the amide group of the highly conserved Gln-125 and hydrogen bonds with Arg-176 by means of two ordered water molecules. Moreover, the pyrimidine ring of thymidine is fixed between Met-128 and T...

Kinetic and crystallographic analyses of wild-type Herpes simplex virus type 1 thymidine kinase (TK(HSV1)) and its Y101F-mutant [TK(HSV1)(Y101F)] acting on the potent antiviral drug 2'-exo-methanocarba-thymidine (MCT) have been performed. The kinetic study reveals a 12-fold K(M) increase for thymidine processed with Y101F as compared to the wild-type TK(HSV1). Furthermore, MCT is a substrate for both wild-type and mutant TK(HSV1). Its binding affinity for TK(HSV1) and TK(HSV1)(Y101F), expressed as K(i), is 11 microM and 51 microM, respectively, whereas the K(i) for human cytosolic thymidine kinase is as high as 1.6 mM, rendering TK(HSV1) a selectivity filter for antiviral activity. Moreover, TK(HSV1)(Y101F) shows a decrease in the quotient of the catalytic efficiency (k(cat)/K(M)) of dT over MCT corresponding to an increased specificity for MCT when compared to the wild-type enzyme. Crystal structures of wild-type and mutant TK(HSV1) in complex with MCT have been determined to resolutions of 1.7 and 2.4 A, respectively. The thymine moiety of MCT binds like the base of dT while the conformationally restricted bicyclo[3.1.0]hexane, mimicking the sugar moiety, assumes a 2'-exo envelope conformation that is flatter than the one observed for the free compound. The hydrogen bond pattern around the sugar-like moiety differs from that of thymidine, revealing the importance of the rigid conformation of MCT with respect to hydrogen bonds. These findings make MCT a lead compound in the design of resistance-repellent drugs for antiviral therapy, and mutant Y101F, in combination with MCT, opens new possibilities for gene therapy.