Feb 15, 2017 - USA, 2Chemical and Biomolecular Engineering, Chemistry, Bioengineering,. UC Berkeley, Berkeley, CA, USA, 3Chemical Sciences Division, ...
Wednesday, February 15, 2017
Posters Protein Structure and Conformation IV 2394-Pos Board B1 Development and Comparison of Enhanced Sampling Methods for Biomolecular Simulation James Lincoff1, Sukanya Sasmal1, Teresa Head-Gordon2,3. 1 Chemical and Biomolecular Engineering, UC Berkeley, Berkeley, CA, USA, 2Chemical and Biomolecular Engineering, Chemistry, Bioengineering, UC Berkeley, Berkeley, CA, USA, 3Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. Molecular dynamics (MD) simulations generate structural ensembles of proteins at atomic-level resolution, and are particularly valuable for studying intrinsically disordered proteins, whose ensembles cannot be fully characterized experimentally. Standard MD is impractical as trajectories have to be run for intractably long times in order to converge to the correct structural distribution. Enhanced sampling methods, of which temperature replica exchange (TREx) is the most common, are used to overcome this sampling problem. However, TREx is also limited due to its very high computational expense for large systems and sub-optimal rate of convergence. We have developed a new enhanced sampling method, temperature cool walking (TCW), that demonstrates marked improvements in both of these areas. Originally developed and tested on a one-dimensional potential, TCW converges to the equilibrium probability distribution of that system more rapidly than optimally scheduled TREx simulations. We have extended TCW to peptides, and codes are now available in OpenMM, an open-source platform for running MD on GPUs. We validate on two test peptides, alanine dipeptide and met-enkephalin, that TCW more rapidly converges to the equilibrium distributions generated by long standard MD trajectories than TREx simulations, at one third the computational cost of TREx. We then apply TCW to the amyloid beta (Ab) family of peptides, known key players in Alzheimer’s disease. On these systems, TCW has 13 % the cost used to generate trajectories of the same length using TREx, and has improved agreement with NMR observables, demonstrating that we have overcome the limitation in sampling of TREx. The computational savings and improved convergence will allow for more ambitious calculations in the future. 2395-Pos Board B2 Structural Characteristics of the RNAse H Domain in HIV-1 Reverse Transcriptase Ryan L. Slack, Naima G. Sharaf, Angela M. Gronenborn, Rieko Ishima. Structural Biology, University of Pittsburgh, Pittsburgh, PA, USA. HIV-1 Reverse Transcriptase (RT) is a multi-functional enzyme responsible for the viral DNA polymerase and ribonuclease activities required for viral replication. RT is encoded as a 66 kDa polypeptide in the Gag-Pol polyprotein and matured to form a heterodimer composed of 66 kDa (p66) and 51 kDa (p51) subunits. The maturation process of RT is believed to entail dimerization of p66 subunits, followed by cleavage of the C-terminal ribonuclease H domain (RNH) in one of the p66 subunits by viral protease (PR), yielding the p51 subunit within the mature heterodimer. In structures of mature RT, the p51-RNH cleavage site in the remaining p66 subunit is buried within the RNH core. Therefore the structural determinants which allow for PR recognition and cleavage of a single p66 subunit in the immature p66 homodimer remain unclear. Thus, we performed solution NMR experiments of the immature p66 homodimer to elucidate the conformation of the RNH domain within this complex. Additional NMR experiments were conducted to investigate the effect of buffer ionic strength on subunit dimerization. Finally, we introduced mutations within the isolated RNH domain to clarify structural characteristics of the cleavage site using NMR and other biophysical methods. (Supported by: the National Institutes of Health P50GM082251 and R01GM105401). 2396-Pos Board B3 Effects of ALS Mutations on Structure, Dynamics, and Function of Ubiquilin-2 Carlos A. Castaneda, Thuy P. Dao. Chemistry and Biology, Syracuse University, Syracuse, NY, USA. Amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases are caused by malfunctions in ubiquitin-mediated protein degradation pathways. Ubiquilin-2 is a multi-domain adaptor protein critical for maintaining protein homeostasis through the ubiquitin-proteasome system, the endoplasmic reticulum-associated protein degradation (ERAD) pathway and autophagy. Mutations in ubiquilin-2 have recently been shown to cause dominant x-linked inheritance of ALS and ALS/dementia. Moreover, wild-type ubiquilin-2 is present in protein inclusions found in patients diagnosed with either familial or
487a
sporadic ALS, further linking ubiquilin-2 to the pathogenesis of ALS. Interestingly, most ALS-linked mutations are localized to the proline-rich repeat (Pxx) region that is unique to ubiquilin-2 and not present in the other members of the ubiquilin family. The consequences of these mutations (specifically to residues P497, P506, P509, P525 and P533) on ubiquilin-2’s structure and function remain unknown. Towards that end, we are using biomolecular NMR spectroscopy to characterize the Pxx region of ubiquilin-2. 13C-detect NMR experiments have allowed for the direct and necessary observation of the prolines as well as aided in resonance assignment for a significant portion of this ubiquilin-2 region. Significantly, even though the Pxx region is intrinisically disordered, we observed sequence-distant perturbations in several ALSlinked mutants. Serendipitously, similar to some other ALS-linked proteins, our ubiquilin-2 constructs exhibit salt and temperature dependent liquid-like phase-separation, a behavior also affected by ALS mutations. We hypothesize that ALS mutations alter the structure and dynamics of the Pxx region. We are working towards modeling the structure of ubiquilin-2 containing the Pxx region, as well as identifying protein-binding surfaces involving the Pxx region. 2397-Pos Board B4 Energy Landscapes of a Mechanical Prion and their Implications for the Molecular Mechanisms of Long-Term Memory Mingchen Chen. Rice University, Houston, TX, USA. Aplysia cytoplasmic polyadenylation element binding (CPEB) pro- tein, a translational regulator that recruits mRNAs and facilitates translation, has been shown to be a key component in the formation of long-term memory. Experimental data show that CPEB exists in at least a low-molecular weight coiledcoil oligomeric form and an amyloid fiber form involving the Q-rich domain (CPEB-Q). Using a coarse-grained energy landscape model, we predict the structures of the low-molecular weight oligomeric form and the dynamics of their transitions to the b-form. Up to the decamer, the oligomeric struc- tures are predicted to be coiled coils. Free energy profiles confirm that the coiled coil is the most stable form for dimers and trimers. The structural transition from a to b is shown to be concentration dependent, with the transition barrier decreasing with increased concentration. We observe that a mechanical pulling force can facil- itate the a-helix to b-sheet (a-to-b) transition by lowering the free energy barrier between the two forms. Interactome analysis of the CPEB protein suggests that its interactions with the cytoskeleton could provide the necessary mechanical force. We propose that, by exerting mechanical forces on CPEB oligomers, an active cytoskele- ton can facilitate fiber formation. This mechanical catalysis makes possible a positive feedback loop that would help localize the formation of CPEB fibers to active synapse areas and mark those synapses for forming a long-term memory after the prion form is established. The functional role of the CPEB helical oligomers in this mechanism carries with it implications for targeting such spe- cies in neurodegenerative diseases. 2398-Pos Board B5 Methyl-Labeling Assisted NMR Structure Determination of a 66 KDA Growth Factor-Receptor Complex Andrew Hinck1, Morkos A. Henen1, Christian Zwieb2, Ravindra Kodali1, Cynthia S. Hinck1. 1 Dept. of Structural Biology, University of Pittsburgh, Pittsburgh, PA, USA, 2 Dept. of Biochemistry, UT Health Sci. Car. at San Antonio, San Antonio, TX, USA. TGF-b1, TGF- b2, and TGF-b3 are 26 kDa disulfide-linked homodimeric signaling proteins. They all signal through the TGF-b type I and type II receptors, yet TGF-b2, which is well known to bind TbRII several-hundred fold more weakly than TGF-b1 and TGF-b3, has an additional requirement for the TGF-b type III receptor (TbRIII), a membrane-anchored non-signaling receptor that potentiates the binding of TbRII. Though it is known that TbRIII has two component domains that bind TGF-b2 non-cooperatively at independent sites, the structure of these domains bound to TGF-b2 and residues responsible for specific binding are not yet known. The objective of this study was to determine the three-dimensional structure of the 66 kDa 2:1 complex formed between the TbRIII C-terminal domain (RIIIC2) and TGF-b2, the structures of which are both known. To obtain the necessary experimental restraints to determine an accurate three-dimensional structure of the complex, the backbone resonances of unbound TGF-b2 were assigned, extended to the sidechain methyls of Ile, Leu, and Val, and confirmed by Ile -> Leu and Leu/Val -> Ile dropout variants. To identify the interface residues, fully deuterated Ile d1 and Leu and Val proS methyl-protonated proteins were prepared and used to identify methyl chemical shift perturbations (CSPs). The results show that the binding site for RIIIc on TGF-b2 lies on the concave surface of the fingers and includes I33, I92, L101. This is consistent with accompanying mutagenesis data, which shows that substitutions that had the greatest effect on binding correspond to residues that were perturbed to the greatest extent in the NMR
