Mapping the Structural and Dynamical Features of Kinesin Motor Domains

Kinesin motor proteins drive intracellular transport by coupling ATP hydrolysis to conformational changes that mediate directed movement along microtubules. Characterizing these distinct conformations and their interconversion mechanism is essential to determining an atomic-level model of kinesin action. Here we report a comprehensive principal component analysis of 114 experimental structures along with the results of conventional and accelerated molecular dynamics simulations that together map the structural dynamics of the kinesin motor domain. All experimental structures were found to reside in one of three distinct conformational clusters (ATP-like, ADP-like and Eg5 inhibitor-bound). These groups differ in the orientation of key functional elements, most notably the microtubule binding α4–α5, loop8 subdomain and α2b-β4-β6-β7 motor domain tip. Group membership was found not to correlate with the nature of the bound nucleotide in a given structure. However, groupings were coincident with distinct neck-linker orientations. Accelerated molecular dynamics simulations of ATP, ADP and nucleotide free Eg5 indicate that all three nucleotide states could sample the major crystallographically observed conformations. Differences in the dynamic coupling of distal sites were also evident. In multiple ATP bound simulations, the neck-linker, loop8 and the α4–α5 subdomain display correlated motions that are absent in ADP bound simulations. Further dissection of these couplings provides evidence for a network of dynamic communication between the active site, microtubule-binding interface and neck-linker via loop7 and loop13. Additional simulations indicate that the mutations G325A and G326A in loop13 reduce the flexibility of these regions and disrupt their couplings. Our combined results indicate that the reported ATP and ADP-like conformations of kinesin are intrinsically accessible regardless of nucleotide state and support a model where neck-linker docking leads to a tighter coupling of the microtubule and nucleotide binding regions. Furthermore, simulations highlight sites critical for large-scale conformational changes and the allosteric coupling between distal functional sites.     

Structural and Functional Insights into the HIV-1 Maturation Inhibitor Binding Pocket

Processing of the Gag precursor protein by the viral protease during particle release triggers virion maturation, an essential step in the virus replication cycle. The first-in-class HIV-1 maturation inhibitor dimethylsuccinyl betulinic acid [PA-457 or bevirimat (BVM)] blocks HIV-1 maturation by inhibiting the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. A structurally distinct molecule, PF-46396, was recently reported to have a similar mode of action to that of BVM. Because of the structural dissimilarity between BVM and PF-46396, we hypothesized that the two compounds might interact differentially with the putative maturation inhibitor-binding pocket in Gag. To test this hypothesis, PF-46396 resistance was selected for in vitro. Resistance mutations were identified in three regions of Gag: around the CA-SP1 cleavage site where BVM resistance maps, at CA amino acid 201, and in the CA major homology region (MHR). The MHR mutants are profoundly PF-46396-dependent in Gag assembly and release and virus replication. The severe defect exhibited by the inhibitor-dependent MHR mutants in the absence of the compound is also corrected by a second-site compensatory change far downstream in SP1, suggesting structural and functional cross-talk between the HIV-1 CA MHR and SP1. When PF-46396 and BVM were both present in infected cells they exhibited mutually antagonistic behavior. Together, these results identify Gag residues that line the maturation inhibitor-binding pocket and suggest that BVM and PF-46396 interact differentially with this putative pocket. These findings provide novel insights into the structure-function relationship between the CA MHR and SP1, two domains of Gag that are critical to both assembly and maturation. The highly conserved nature of the MHR across all orthoretroviridae suggests that these findings will be broadly relevant to retroviral assembly. Finally, the results presented here provide a framework for increased structural understanding of HIV-1 maturation inhibitor activity.     

Mutations in DNA Binding and Transactivation Domains Affect the Dynamics of Parvovirus NS1 Protein

The multifunctional replication protein of autonomous parvoviruses, NS1, is vital for viral genome replication and for the control of viral protein production. Two DNA-interacting domains of NS1, the N-terminal and helicase domains, are necessary for these functions. In addition, the N and C termini of NS1 are required for activation of viral promoter P38. By comparison with the structural and biochemical data from other parvoviruses, we identified potential DNA-interacting amino acid residues from canine parvovirus NS1. The role of the identified amino acids in NS1 binding dynamics was studied by mutagenesis, fluorescence recovery after photobleaching, and computer simulations. Mutations in the predicted DNA-interacting amino acids of the N-terminal and helicase domains increased the intranuclear binding dynamics of NS1 dramatically. A substantial increase in binding dynamics was also observed for NS1 mutants that targeted the metal ion coordination site in the N terminus. Interestingly, contrary to other mutants, deletion of the C terminus resulted in slower binding dynamics of NS1. P38 transactivation was severely reduced in both N-terminal DNA recognition and in C-terminal deletion mutants. These data suggest that the intranuclear dynamics of NS1 are largely characterized by its sequence-specific and -nonspecific binding to double-stranded DNA. Moreover, binding of NS1 is equally dependent on the N-terminal domain and conserved β-loop of the helicase domain.     

Structural Insight into Chromatin Recognition by Multiple Domains of the Tumor Suppressor RBBP1

Retinoblastoma-binding protein 1 (RBBP1) is involved in gene regulation, epigenetic regulation, and disease processes. RBBP1 contains five domains with DNA-binding or histone-binding activities, but how RBBP1 specifically recognizes chromatin is still unknown. An AT-rich interaction domain (ARID) in RBBP1 was proposed to be the key region for DNA-binding and gene suppression. Here, we first determined the solution structure of a tandem PWWP-ARID domain mutant of RBBP1 after deletion of a long flexible acidic loop L12 in the ARID domain. NMR titration results indicated that the ARID domain interacts with DNA with no GC- or AT-rich preference. Surprisingly, we found that the loop L12 binds to the DNA-binding region of the ARID domain as a DNA mimic and inhibits DNA binding. The loop L12 can also bind weakly to the Tudor and chromobarrel domains of RBBP1, but binds more strongly to the DNA-binding region of the histone H2A-H2B heterodimer. Furthermore, both the loop L12 and DNA can enhance the binding of the chromobarrel domain to H3K4me3 and H4K20me3. Based on these results, we propose a model of chromatin recognition by RBBP1, which highlights the unexpected multiple key roles of the disordered acidic loop L12 in the specific binding of RBBP1 to chromatin.     

New Insights into the Role of DNA Shape on Its Recognition by p53 Proteins

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Structural and Dynamical Insights on HLA-DR2 Complexes That Confer Susceptibility to Multiple Sclerosis in Sardinia: A Molecular Dynamics Simulation Study

Sardinia is a major Island in the Mediterranean with a high incidence of multiple sclerosis, a chronic autoimmune inflammatory disease of the central nervous system. Disease susceptibility in Sardinian population has been associated with five alleles of major histocompatibility complex (MHC) class II DRB1 gene. We performed 120 ns of molecular dynamics simulation on one predisposing and one protective alleles, unbound and in complex with the two relevant peptides: Myelin Basic Protein and Epstein Barr Virus derived peptide. In particular we focused on the MHC peptide binding groove dynamics. The predisposing allele was found to form a stable complex with both the peptides, while the protective allele displayed stability only when bound with myelin peptide. The local flexibility of the MHC was probed dividing the binding groove into four compartments covering the well known peptide anchoring pockets. The predisposing allele in the first half cleft exhibits a narrower and more rigid groove conformation in the presence of myelin peptide. The protective allele shows a similar behavior, while in the second half cleft it displays a narrower and more flexible groove conformation in the presence of viral peptide. We further characterized these dynamical differences by evaluating H-bonds, hydrophobic and stacking interaction networks, finding striking similarities with super-type patterns emerging in other autoimmune diseases. The protective allele shows a defined preferential binding to myelin peptide, as confirmed by binding free energy calculations. All together, we believe the presented molecular analysis could help to design experimental assays, supports the molecular mimicry hypothesis and suggests that propensity to multiple sclerosis in Sardinia could be partly linked to distinct peptide-MHC interaction and binding characteristics of the antigen presentation mechanism.     

Structural Insights into the Nucleotide-Binding Domains of the P1B-type ATPases HMA6 and HMA8 from Arabidopsis thaliana

Copper is a crucial ion in cells, but needs to be closely controlled due to its toxic potential and ability to catalyse the formation of radicals. In chloroplasts, an important step for the proper functioning of the photosynthetic electron transfer chain is the delivery of copper to plastocyanin in the thylakoid lumen. The main route for copper transport to the thylakoid lumen is driven by two P_IB-type ATPases, Heavy Metal ATPase 6 (HMA6) and HMA8, located in the inner membrane of the chloroplast envelope and in the thylakoid membrane, respectively. Here, the crystal structures of the nucleotide binding domain of HMA6 and HMA8 from Arabidopsis thaliana are reported at 1.5Å and 1.75Å resolution, respectively, providing the first structural information on plants Cu⁺-ATPases. The structures reveal a compact domain, with two short helices on both sides of a twisted beta-sheet. A double mutant, aiding in the crystallization, provides a new crystal contact, but also avoids an internal clash highlighting the benefits of construct modifications. Finally, the histidine in the HP motif of the isolated domains, unable to bind ATP, shows a side chain conformation distinct from nucleotide bound structures.     

Insights into the Structural Dynamics of Nucleocytoplasmic Transport of tRNA by Exportin-t

Exportin-t (Xpot) transports mature 5′- and 3′-end processed tRNA from the nucleus to the cytoplasm by associating with a small G-protein Ran (RAs-related nuclear protein), in the nucleus. The release of tRNA in cytoplasm involves RanGTP hydrolysis. Despite the availability of crystal structures of nuclear and cytosolic forms of Xpot, the molecular details regarding the sequential events leading to tRNA release and subsequent conformational changes occurring in Xpot remain unknown. We have performed a combination of classical all-atom and accelerated molecular dynamics simulations on a set of complexes involving Xpot to study a range of features including conformational flexibility of free and cargo-bound Xpot and functionally critical contacts between Xpot and its cargo. The systems investigated include free Xpot and its different complexes, bound either to Ran (GTP/GDP) or tRNA or both. This approach provided a statistically reliable estimate of structural dynamics of Xpot after cargo release. The mechanistic basis for Xpot opening after cargo release has been explained in terms of dynamic structural hinges, about which neighboring region could be displaced to facilitate the nuclear to cytosolic state transition. Post-RanGTP hydrolysis, a cascade of events including local conformational change in RanGTP and loss of critical contacts at Xpot/tRNA interface suggest factors responsible for eventual release of tRNA. The level of flexibility in different Xpot complexes varied depending on the arrangement of individual HEAT repeats. Current study provides one of the most comprehensive and robust analysis carried out on this protein using molecular dynamics schemes.