Biomedical subjects

F Alber

Publications and source records attributed to F Alber.

6 recordsLinked to original sources

PubMed2001

Ser133 phosphate-KIX interactions in the CREB-CBP complex: an ab initio molecular dynamics study.

Cyclic AMP response element binding protein (CREB) is involved in activation of transcriptional DNA machinery by binding to the coactivator CREB-binding protein (CBP). The interactions between CREB serine phosphate (pSer133) and specific CBP residues (Tyr658 and Lys662) play a crucial role for the thermodynamic stability of the CREB-CBP complex. Here we use ab initio methods to investigate the dynamics and energetics of a relatively large, fully hydrated model complex representing pSer133 and its counterparts of the CBP domain. The calculations suggest that: (1) key contributions to the stabilization of the complex arise not only from electrostatics (as previously proposed) but also from a previously unrecognized "low-barrier hydrogen bond" between pSer133 and Lys662; (2) hydration plays a crucial role for the stabilization of the phosphate charge; (3) formation of the complex involves a significant degree of reorganization of the electronic charge density.

Binding Sites↗

PubMed2000

An analysis of the helix-to-strand transition between peptides with identical sequence.

An analysis of peptide segments with identical sequence but that differ significantly in structure was performed over non-redundant databases of protein structures. We focus on those peptides, which fold into an alpha-helix in one protein but a beta-strand in another. While the study shows that many such structurally ambivalent peptides contain amino acids with a strong helical preference collocated with amino acids with a strong strand preference, the results overwhelmingly indicate that the peptide's environment ultimately dictates its structure. Furthermore, the first naturally occurring structurally ambivalent nonapeptide from evolutionary unrelated proteins is described, highlighting the intrinsic plasticity of peptide sequences. We even find seven proteins that show structural ambivalence under different conditions. Finally, a computer algorithm has been implemented to identify regions in a given sequence where secondary structure prediction programs are likely to make serious mispredictions.

Algorithms↗

PubMed2000

Ab initio molecular dynamics studies on HIV-1 reverse transcriptase triphosphate binding site: implications for nucleoside-analog drug resistance.

Quantum-chemical methods are used to shed light on the functional role of residues involved in the resistance of HIV-1 reverse transcriptase against nucleoside-analog drugs. Ab initio molecular dynamics simulations are carried out for models representing the adduct between the triphosphate substrate and the nucleoside binding site. The triphosphate is considered either deprotonated or protonated at the gamma-position. Although the protonated form already experiences large rearrangements in the ps time scale, the fully deprotonated state exhibits a previously unrecognized low-barrier hydrogen bond between Lys65 and gamma-phosphate. Absence of this interaction in Lys65-->Arg HIV-1 RT might play a prominent role in the resistance of this mutant for nucleoside analogs (Gu Z et al., 1994b, Antimicrob Agents Chemother 38:275-281; Zhang D et al., 1994, Antimicrob Agents Chemother 38:282-287). Water molecules present in the active site, not detected in the X-ray structure, form a complex H-bond network. Among these waters, one may be crucial for substrate recognition as it bridges Gln151 and Arg72 with the beta-phosphate. Absence of this stabilizing interaction in Gln151-->Met HIV-1 RT mutant may be a key factor for the known drug resistance of this mutant toward dideoxy-type drugs and AZT (Shirasaka T et al., 1995, Proc Natl Acad Sci USA 92:2398-2402: Iversen AK et al., 1996, J Virol 70:1086-1090).

Amino Acid Substitution↗

PubMed1999

Substrate diversity of herpes simplex virus thymidine kinase. Impact Of the kinematics of the enzyme.

Herpes simplex virus type 1 (HSV 1) thymidine kinase (TK) exhibits an extensive substrate diversity for nucleobases and sugar moieties, in contrast to other TKs. This substrate diversity is the crucial molecular basis of selective antiviral and suicide gene therapy. The mechanisms of substrate binding of HSV 1 TK were studied by means of site-directed mutagenesis combined with isothermal calorimetric measurements and guided by theoretical calculations and sequence comparison. The results show the link between the exceptionally broad substrate diversity of HSV 1 TK and the presence of structural features such as the residue triad His-58/Met-128/Tyr-172. The mutation of Met-128 into a Phe and the double mutant M128F/Y172F result in mutants that have lost their activity. However, by exchanging His to form the triple mutant H58L/M128F/Y172F, the enzyme regains activity. Strikingly, this triple mutant becomes resistant toward acyclovir. Furthermore, we give evidence for the importance of Glu-225 of the flexible LID region for the catalytic reaction. The data presented give new insights to understand mechanisms ruling substrate diversity and thus are crucial for both the development of new antiviral drugs and engineering of mutant TKs apt to accept novel substrate analogs for gene therapeutic approaches.

Amino Acid Sequence↗

PubMed1998

Density functional studies on herpes simplex virus type 1 thymidine kinase-substrate interactions: the role of Tyr-172 and Met-128 in thymine fixation.

The enzyme herpes simplex virus type 1 thymidine kinase (HSV1 TK) salvages thymidine into the DNA metabolism of the virus. In the active site, the thymine ring of the nucleoside binds in a pocket, formed by two residues, Tyr-172 and Met-128, in a sandwich-type orientation. To investigate the nature of the thymine-enzyme pocket interactions, we have carried out density functional theory calculations with gradient-corrected exchange-correlation functionals of models of the thymine-HSV1 TK adduct. Our calculations indicate that the role of Met-128 in the substrate fixation is purely steric and hydrophobic, while the substrate-Tyr-172 interaction is essentially electrostatic in nature. These findings are completely consistent with the available catalytic properties of mutants on the 128 position.

Adenosine Triphosphate↗

PubMedDate not supplied

Integrated homology modelling and X-ray study of herpes simplex virus I thymidine kinase: a case study.

Knowledge-based homology modelling together with site-directed mutagenesis, epitope and conformational mapping is an approach to predict the structures of proteins and for the rational design of new drugs. In this study we present how this procedure has been applied to model the structure of herpes simplex virus type 1 thymidine kinase (HSV1 TK, HSV1 ATP-thymidine-5'-phosphotransferase, EC 2.7.1.21). We have used, and evaluated, several secondary structure prediction methods, such as the classical one based on Chou and Fastman algorithm, neural networks using the Kabsch and Sander classification, and the PRISM method. We have validated the algorithms by applying them to the porcine adenylate kinase (ADK), whose three-dimensional structure is known and that has been used for the alignment of the TKs as well. The resulting first model of HSV1-TK consisted of the first beta-strand connected to the phosphate binding loop and its subsequent alpha-helix, the fourth beta-strand connected to the conserved FDRH sequence and two alpha-helix with basic amino acids. The 3D structure was built using the X-ray structure of ADK as template and following the general procedure for homology modelling. We extended the model by means of COMPOSER, an automatic process for protein modelling. Site-directed mutagenesis was used to experimentally verify the predicted active-site model of HSV1-TK. The data measured in our lab and by others support the suggestion that the FDRH motif is part of the active site and plays an important role in the phosphorylation of substrates. The structure of HSV1 TK, recently solved in collaboration with Prof. G. Schulz at 2.7 A resolution, includes 284 of 343 residues of the N-terminal truncated TK. The secondary structures could be clearly assigned and fitted to the density. The comparison between crystallographically determined structure and the model shows that nearly 70% of the HSV1 TK structure has been correctly modelled by the described integrated approach to knowledge based ligand protein complex structure prediction. This indicate that computer assisted methods, combined with "manual" correction both for alignment and 3D construction are useful and can be successful.

Crystallography, X-Ray↗