A human transcription factor in search mode

Kevin Hauser1,2, Bernard Essuman3, Yiqing He4, Evangelos Coutsias1,5, Miguel Garcia-Diaz6 and Carlos Simmerling1,2,

1Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY 11794, USA. 2Department of Chemistry, Stony Brook University, Stony Brook, NY 11794, USA. 3Suffolk Community College, Selden, NY 11784, USA. 4Great Neck South High School, Great Neck, NY 11023, USA. 5Department of Applied Mathematics and Statistics, Stony Brook University, Stony Brook, NY 11794, USA. 6Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA. *To whom correspondence should be addressed.


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Abstract

Transcription factors (TF) can change shape to bind and recognize DNA, shifting the energy landscape from a weak binding, rapid search mode to a higher affinity recognition mode. However, the mechanism(s) driving this conformational change remains unresolved and in most cases high-resolution structures of the non-specific complexes are unavailable. Here, we investigate the conformational switch of the human mitochondrial transcription termination factor MTERF1, which has a modular, superhelical topology complementary to DNA. Our goal was to characterize the details of the non-specific search mode to complement the crystal structure of the specific binding complex, providing a basis for understanding the recognition mechanism. In the specific complex, MTERF1 binds a significantly distorted and unwound DNA structure, exhibiting a protein conformation incompatible with binding to B-form DNA. In contrast, our simulations of apo MTERF1 revealed significant flexibility, sampling structures with superhelical pitch and radius complementary to the major groove of B-DNA. Docking these structures to B-DNA followed by unrestrained MD simulations led to a stable complex in which MTERF1 was observed to undergo spontaneous diffusion on the DNA. Overall, the data support an MTERF1-DNA binding and recognition mechanism driven by intrinsic dynamics of the MTERF1 superhelical topology. 


 

Base Flipping by MTERF1 Can Accommodate Multiple Conformations and Occurs in a Stepwise Fashion

James Byrnes1*, Kevin Hauser2*, Leah Norona1, Edison Mejia1, Carlos Simmerling3, Miguel Garcia-Diaz4 

  • 1Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA. 2Department of Chemistry, Stony Brook University, Stony Brook, NY 11794, USA. 3Department of Chemistry, Stony Brook University, Stony Brook, NY 11794, USA; Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY 11794, USA. 4Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA. 
  • *Co-first authors

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Abstract

Human mitochondrial transcription termination occurs within the leu-tRNA gene and is mediated by the DNA binding protein MTERF1. The crystal structure of MTERF1 bound to the canonical termination sequence reveals a rare base flipping event that involves the eversion of three nucleotides. These nucleotides are stabilized by stacking interactions with three MTERF1 residues, which are essential not only for base flipping but also for termination activity. To further understand the mechanism of base flipping, we examined each of the individual stacking interactions in structural, energetic and functional detail. Individual substitutions of Arg162, Tyr288 and Phe243 have revealed unequal contributions to overall termination activity. Furthermore, our work identifies an important role for Phe322 in the base flipping mechanism and we demonstrate how Phe322 and Phe243 are important for coupling base flipping between the heavy and light strand DNA chains. We propose a stepwise model for the base flipping process that recapitulates our observations. Finally, we show that MTERF1 has the ability to accommodate alternate active conformations. The adaptability of base flipping has implications for MTERF1 function and for the putative function of MTERF1 at alternative binding sites in human mitochondria.

ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB

James A. Maier†§, Carmenza Martinez‡§, Koushik Kasavajhala‡§, Lauren Wickstrom†, Kevin E. Hauser‡§, and Carlos Simmerling*†‡§

†Graduate Program in Biochemistry and Structural Biology, ‡Department of Chemistry, and §Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, New York 11794, United States


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Abstract

Molecular mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Average errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a physically motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reproduction of NMR χ1 scalar coupling measurements for proteins in solution. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.