Biomedical subjects

Yongping Pan

Publications and source records attributed to Yongping Pan.

14 recordsLinked to original sources

PubMed2006

Structural basis for p53 binding-induced DNA bending.

Specific p53 binding-induced DNA bending has important biological implications such as transcription activation. However, the detailed structures of the bent DNA and the p53-DNA complex are still unavailable, hampering our understanding of the mechanism for p53-induced DNA bending and its consequent biological significance. To gain insight into the p53 binding-induced DNA bending, we performed molecular dynamics simulations on DNA segments with the consensus sequence for p53-specific binding, half site DNA-p53 complexes, and full site DNA-p53 complexes. We show that each DNA-bound p53 core domain caused a local DNA conformational change within the quarter site; upon the binding of the p53 dimer, there was an apparent DNA bending at the center of the half site; when bound with two p53 dimers, the full site DNAs with two different sequences bent 20 and 35 degrees, respectively. These results are in agreement with experimental observations. Our simulations demonstrate that the two p53 dimers favored a staggered conformation in which they make favorable interactions at the interface. This dimer-dimer interface organization necessitated conformational changes in the DNA, leading to the bending at the center of the full site, which in turn is dependent on the DNA sequence. Overall, our results provide the detailed atomic model for the DNA-p53 tetramer complex and delineate the roles of DNA-p53, p53 dimer-dimer interactions, and DNA sequence in specific p53 binding-induced DNA conformational changes.

Binding Sites↗

PubMed2006

Importance of context in protein folding: secondary structural propensities versus tertiary contact-assisted secondary structure formation.

Molecular dynamics simulations can be used to reveal the detailed conformational behaviors of peptides and proteins. By comparing fragment and full-length protein simulations, we can investigate the role of each peptide segment in the folding process. Here, we take advantage of information regarding the helix formation process from our previous simulations of barnase and protein A as well as new simulations of four helical fragments from these proteins at three different temperatures, starting with both helical and extended structures. Segments with high helical propensity began the folding process by tethering the chain through side chain interactions involving either polar interactions, such as salt bridges, or hydrophobic staples. These tethers were frequently nonnative (i.e., not i --> i + 4 spacing) and provided a scaffold for other residues, thereby limiting the conformational search. The helical structure then propagated on both sides of the tether. Segments with low stability and propensity formed later in the folding process and utilized contacts with other portions of the protein when folding. These helices formed via a tertiary contact-assisted mechanism, primarily via hydrophobic contacts between residues distant in sequence. Thus, segments with different helical propensities appear to play different roles during protein folding. Furthermore, the active role of nonlocal side chains in helix formation highlights why we must move beyond simple hierarchical models of protein folding.

Amino Acid Sequence↗

PubMed2006

Comparison of the human and worm p53 structures suggests a way for enhancing stability.

Maintaining the native conformation is essential for the proper function of tumor suppressor protein p53. However, p53 is a low-stability protein that can easily lose its function upon structural perturbations such as those resulting from missense mutations, leading to the development of cancer. Therefore, it is important to develop strategies to design stable p53 which still maintains its normal function. Here, we compare the stabilities of the human and worm p53 core domains using molecular dynamics simulations. We find that the worm p53 is significantly more stable than the human form. Detailed analysis of the structural fluctuations shows that the stability difference lies in the peripheral structural motifs that contrast in their structural features and flexibility. The most dramatic difference in stability originates from loop L1, from the turn between helix H1 and beta-strand S5, and from the turn that connects beta-strands S7 and S8. Structural analysis shows significant differences for these motifs between the two proteins. Loop L1 lacks secondary structure, and the turns between helix H1 and strand S5 and between strands S7 and S8 are much longer in the human form p53. On the basis of these differences, we designed a mutant by shortening the turn between strands S7 and S8 to enhance the stability. Surprisingly, this mutant was very stable when probed by molecular dynamics simulations. In addition, the stabilization was not localized in the turn region. Loop L1 was also significantly stabilized. Our results show that stabilizing peripheral structural motifs can greatly enhance the stability of the p53 core domain and therefore is likely to be a viable alternative in the development of stable p53. In addition, loop- or turn-related mutants with different stabilities may also be used to probe the relationship between function, a particular structural motif, and its flexibility.

Animals↗

PubMed2006

"Similarity trap" in protein-protein interactions could be carcinogenic: simulations of p53 core domain complexed with 53BP1 and BRCA1 BRCT domains.

Similar binding sites often imply similar protein-protein interactions and similar functions; however, similar binding sites may also constitute traps for nonfunctional associations. How are similar sites distinguished to prevent misassociations? BRCT domains from breast cancer-susceptibility gene product BRCA1 and protein 53BP1 have similar structures yet different binding behaviors with p53 core domain. 53BP1-BRCT domain forms a stable complex with p53. In contrast, BRCA1-p53 interaction is weak or other mechanisms operate. To delineate the difference, we designed 13 BRCA1-BRCT mutants and computationally investigated the structural and stability changes compared to the experimental p53-53BP1 structure. Interestingly, of the 13, the 2 mutations that are cancerous and involve nonconserved residues are those that enforced p53 core domain binding with BRCA1-BRCT in a way similar to p53-53BP1 binding. Hence, falling into the "similarity trap" may disrupt normal BRCA1 and p53 functions. Our results illustrate how this trap is avoided in the native state.

Amino Acid Sequence↗

PubMed2005

CD4 binding partially locks the bridging sheet in gp120 but leaves the beta2/3 strands flexible.

The structure of the free form HIV gp120, critical for therapeutic agent development, is unavailable due to its high flexibility. Previous thermodynamic data, structural analysis and simulation results have suggested a large conformational change in the core domain upon CD4 binding. The bridging sheet, which consists of four beta-strands with beta20/21 nestling against the inner/outer domains and beta2/3 facing outward, more exposed to the solvent, was proposed to be unfolded in the native state. In order to test this proposition and to characterize the native conformations, we performed potential mean force (PMF) molecular dynamics (MD) simulations on the CD4-bound crystal structure. We pushed the bridging sheet away from the inner and outer domain to explore the accessible conformational space for the bridging sheet. In addition, we performed conventional MD simulations on structures with the bridging sheet partially unfolded to investigate the stability of the association between the inner and outer domains. Based on the free energy profiles, we find that the whole bridging sheet is unlikely to unfold without other concurrent conformational changes. On the other hand, the partial bridging sheet, beta strands 2/3, can switch its conformation from the folded to the unfolded state. Furthermore, relaxation of conformation with partially unfolded bridging sheet through MD simulations leads to a conformation with beta strands 20/21 quickly re-anchoring against the inner and outer domains. Such a conformation, although lacking some of the hydrophobic interactions present in the CD4-bound structure, displayed high stability as further indicated by other restrained MD simulations. The relevance of this conformation to the free form structure and the pathway for conformational change from the free form to the CD4-bound structure is discussed in detail in light of the available unliganded SIV gp120 crystal structure.

Amino Acid Motifs↗

PubMed2005

Comparison of the protein-protein interfaces in the p53-DNA crystal structures: towards elucidation of the biological interface.

p53, the tumor suppressor protein, functions as a dimer of dimers. However, how the tetramer binds to the DNA is still an open question. In the crystal structure, three copies of the p53 monomers (containing chains A, B, and C) were crystallized with the DNA-consensus element. Although the structure provides crucial data on the p53-DNA contacts, the active oligomeric state is unclear because the two dimeric (A-B and B-C) interfaces present in the crystal cannot both exist in the tetramer. Here, we address the question of which of these two dimeric interfaces may be more biologically relevant. We analyze the sequence and structural properties of the p53-p53 dimeric interfaces and carry out extensive molecular dynamics simulations of the crystal structures of the human and mouse p53 dimers. We find that the A-B interface residues are more conserved than those of the B-C. Molecular dynamics simulations show that the A-B interface can provide a stable DNA-binding motif in the dimeric state, unlike B-C. Our results indicate that the interface between chains A-B in the p53-DNA complex constitutes a better candidate for a stable biological interface, whereas the B-C interface is more likely to be due to crystal packing. Thus, they have significant implications toward our understanding of DNA binding by p53 as well as p53-mediated interactions with other proteins.

Amino Acid Motifs↗

PubMed2005

In the quest for stable rescuing mutants of p53: computational mutagenesis of flexible loop L1.

p53 is a protein with marginal stability. Its transcriptional functions are often inactivated by single missense mutations, shown to be associated with half of all human cancers. Here, we aim to design stable functional p53 mutants. We target loop L1, one of the most mobile structural motifs in the p53 core domain (p53C). Specifically, we selected Ser116 in the middle of loop L1 and mutated it to 14 other amino acids. All resulting mutants were subjected to molecular dynamics simulations, revealing a wide spectrum of stabilities. Among these, mutant S116M displayed a remarkable stability, with a structural deviation comparable to that of the experimental quadruple mutant M133L/V203A/N239Y/N268D that is thermodynamically more stable than that of the wild type by 2.6 kcal/mol. Structural analysis showed that the high stability of the S116M mutant was indeed due to the preservation of the p53C loop L1 conformation and the reduction of mobility in that region. The differential stabilities conferred by the single mutations are rationalized based on the geometries and chemical properties of the side chains introduced into this site. Linearity (i.e., nonbranched), moderate size, and balanced hydrophobic and hydrophilic properties of the side chain are crucial to the stabilizing effect of the residue substitutions.

Amino Acid Substitution↗

PubMed2005

Conformational determinants of tandem GU mismatches in RNA: insights from molecular dynamics simulations and quantum mechanical calculations.

Structure and energetic properties of base pair mismatches in duplex RNA have been the focus of numerous investigations due to their role in many important biological functions. Such efforts have contributed to the development of models for secondary structure prediction of RNA, including the nearest-neighbor model. In RNA duplexes containing GU mismatches, 5'-GU-3' tandem mismatches have a different thermodynamic stability than 5'-UG-3' mismatches. In addition, 5'-GU-3' mismatches in some sequence contexts do not follow the nearest-neighbor model for stability. To characterize the underlying atomic forces that determine the structural and thermodynamic properties of GU tandem mismatches, molecular dynamics (MD) simulations were performed on a series of 5'-GU-3' and 5'-UG-3' duplexes in different sequence contexts. Overall, the MD-derived structural models agree well with experimental data, including local deviations in base step helicoidal parameters in the region of the GU mismatches and the model where duplex stability is associated with the pattern of GU hydrogen bonding. Further analysis of the simulations, validated by data from quantum mechanical calculations, suggests that the experimentally observed differences in thermodynamic stability are dominated by GG interstrand followed by GU intrastrand base stacking interactions that dictate the one versus two hydrogen bonding scenarios for the GU pairs. In addition, the inability of 5'-GU-3' mismatches in different sequence contexts to all fit into the nearest-neighbor model is indicated to be associated with interactions of the central four base pairs with the surrounding base pairs. The results emphasize the role of GG and GU stacking interactions on the structure and thermodynamics of GU mismatches in RNA.

Base Pair Mismatch↗

PubMed2005

The contribution of the Trp/Met/Phe residues to physical interactions of p53 with cellular proteins.

Dynamic molecular interaction networks underlie biological phenomena. Among the many genes which are involved, p53 plays a central role in networks controlling cellular life and death. It not only operates as a tumor suppressor, but also helps regulate hundreds of genes in response to various types of stress. To accomplish these functions as a guardian of the genome, p53 interacts extensively with both nucleic acids and proteins. This paper examines the physical interfaces of the p53 protein with cellular proteins. Previously, in the analysis of the structures of protein-protein complexes, we have observed that amino acids Trp, Met and Phe are important for protein-protein interactions in general. Here we show that these residues are critical for the many functions of p53. Several clusters of the Trp/Met/Phe residues are involved in the p53 protein-protein interactions. Phe19/Trp23 in the TA1 region extensively binds to the transcriptional factors and the MDM2 protein. Trp53/Phe54 in the TA2 region is crucial for transactivation and DNA replication. Met243 in the core domain interacts with 53BP1, 53BP2 and Rad 51 proteins. Met384/Phe385 in the C-terminal region interacts with the S100B protein and the Bromodomain of the CBP protein. Thus, these residues may assist in elucidating the p53 interactions when structural data are not available.

Biophysics↗

PubMed2004

Identification and characterization of small molecule inhibitors of the calcium-dependent S100B-p53 tumor suppressor interaction.

The binding of S100B to p53 down-regulates wild-type p53 tumor suppressor activity in cancer cells such as malignant melanoma, so a search for small molecules that bind S100B and prevent S100B-p53 complex formation was undertaken. Chemical databases were computationally searched for potential inhibitors of S100B, and 60 compounds were selected for testing on the basis of energy scoring, commercial availability, and chemical similarity clustering. Seven of these compounds bound to S100B as determined by steady state fluorescence spectroscopy (1.0 microM < or = K(D) < or = 120 microM) and five inhibited the growth of primary malignant melanoma cells (C8146A) at comparable concentrations (1.0 microM < or = IC(50) < or = 50 microM). Additionally, saturation transfer difference (STD) NMR experiments confirmed binding and qualitatively identified protons from the small molecule at the small molecule-S100B interface. Heteronuclear single quantum coherence (HSQC) NMR titrations indicate that these compounds interact with the p53 binding site on S100B. An NMR-docked model of one such inhibitor, pentamidine, bound to Ca(2+)-loaded S100B was calculated using intermolecular NOE data between S100B and the drug, and indicates that pentamidine binds into the p53 binding site on S100B defined by helices 3 and 4 and loop 2 (termed the hinge region).

Antineoplastic Agents↗

PubMed2004

Characterization of the conformational state and flexibility of HIV-1 glycoprotein gp120 core domain.

gp120 is key to the human immunodeficiency virus type 1 viral cell entry. Knowledge of the detailed conformational states of gp120 is crucial to intervention, yet the unbound form is still resistant to structural characterization probably because of its flexibility. Toward this goal, we performed molecular dynamics simulations on the wild type gp120 core domain extracted from its ternary crystal structure and on a modeled mutant, S375W, that experimentally has a significantly different phenotype from the wild type. Although the mutant retained a bound-like conformation, the wild type drifted to a different conformational state. The wild type beta strands 2 and 3 of the bridging sheet were very mobile and partially unfolded, and the organization among the inner and outer domains and beta strands 20 and 21 of the bridging sheet, near the mutation site, was more open than in the bound form, although the overall structure was maintained. These differences were apparently a result of the strengthening of the hydrophobic core in the mutant. This stabilization further explains the experimentally significantly different thermodynamic properties between the wild type and the mutant. Taken together, our results suggest that the free form, although different from the bound state, shares many of the bound structural features. The observed loss of freedom near the binding site, rather than the previously hypothesized more dramatic conformational transition from the unbound to the bound state, appears to be the major contributor to the large entropy cost for the CD4 binding to the wild type.

Amino Acid Motifs↗

PubMed2003

Altered structural fluctuations in duplex RNA versus DNA: a conformational switch involving base pair opening.

DNA and RNA are known to have different structural properties. In the present study, molecular dynamics (MD) simulations on a series of RNA and DNA duplexes indicate differential structural flexibility for the two classes of oligonucleotides. In duplex RNA, multiple base pairs experienced local opening events into the major groove on the nanosecond time scale, while such events were not observed in the DNA simulations. Three factors are indicated to be responsible for the base opening events in RNA: solvent-base interactions, 2'OH(n)-O4'(n+1) intra-strand hydrogen bonding, and enhanced rigid body motion of RNA at the nucleoside level. Water molecules in the major groove of RNA contribute to initiation of base pair opening. Stabilization of the base pair open state is due to a 'conformational switch' comprised of 2'OH(n)-O4'(n+1) hydrogen bonding and a rigid body motion of the nucleoside moiety in RNA. This rigid body motion is associated with decreased flexibility of the glycosyl linkage and sugar moieties in A-form structures. The observed opening rates in RNA are consistent with the imino proton exchange experiments for AU base pairs, although not for GC base pairs, while structural and flexibility changes associated with the proposed conformational switch are consistent with survey data of RNA and DNA crystal structures. The possible relevance of base pair opening events in RNA to its many biological functions is discussed.

Base Pairing↗

PubMed1997

Characterization of Low-Barrier Hydrogen Bonds. 5. Microsolvation of Enol-Enolate. An ab Initio and DFT Investigation.

Hartree-Fock, Møller-Plesset, and DFT calculations have been carried out using the 6-31+G(d,p) basis set to study the effect of microsolvation on the strength of a representative low-barrier hydrogen bond. In the gas phase, the hydrogen bond formed between vinyl alcohol (enol) and the corresponding oxyanion (enolate anion) is approximately 30 kcal/mol, with a calculated energy barrier for proton transfer from the enol to the enolate anion that is lower than the zero-point vibrational energy resonant in the system. When both the enol and the enolate anion are microsolvated, by one water molecule each, the resulting hydrogen bond is actually increased in strength slightly. When the microsolvation is asymmetrical, however, so as to cause a mismatch in the pK(a) values of the hydrogen-bond donor and hydrogen-bond acceptor, the resulting H-bond is weakened by approximately 4 kcal/mol. These results suggest that small amounts of interstitial water in enzyme active sites may not preclude the existence or importance of low-barrier hydrogen bonds in such biological catalysts.

Journal Article↗

PubMedDate not supplied

Consideration of molecular weight during compound selection in virtual target-based database screening.

Virtual database screening allows for millions of chemical compounds to be computationally selected based on structural complimentary to known inhibitors or to a target binding site on a biological macromolecule. Compound selection in virtual database screening when targeting a biological macromolecule is typically based on the interaction energy between the chemical compound and the target macromolecule. In the present study it is shown that this approach is biased toward the selection of high molecular weight compounds due to the contribution of the compound size to the energy score. To account for molecular weight during energy based screening, we propose normalization strategies based on the total number of heavy atoms in the chemical compounds being screened. This approach is computationally efficient and produces molecular weight distributions of selected compounds that can be selected to be (1) lower than that of the original database used in the virtual screening, which may be desirable for selection of leadlike compounds or (2) similar to that of the original database, which may be desirable for the selection of drug-like compounds. By eliminating the bias in target-based database screening toward higher molecular weight compounds it is anticipated that the proposed procedure will enhance the success rate of computer-aided drug design.

Computer-Aided Design↗