skip to main content


Title: Exploring the folding energy landscapes of heme proteins using a hybrid AWSEM-heme model
Abstract

Heme is an active center in many proteins. Here we explore computationally the role of heme in protein folding and protein structure. We model heme proteins using a hybrid model employing the AWSEM Hamiltonian, a coarse-grained forcefield for the protein chain along with AMBER, an all-atom forcefield for the heme. We carefully designed transferable force fields that model the interactions between the protein and the heme. The types of protein–ligand interactions in the hybrid model include thioester covalent bonds, coordinated covalent bonds, hydrogen bonds, and electrostatics. We explore the influence of different types of hemes (heme b and heme c) on folding and structure prediction. Including both types of heme improves the quality of protein structure predictions. The free energy landscape shows that both types of heme can act as nucleation sites for protein folding and stabilize the protein folded state. In binding the heme, coordinated covalent bonds and thioester covalent bonds for heme c drive the heme toward the native pocket. The electrostatics also facilitates the search for the binding site.

 
more » « less
Award ID(s):
2019745
NSF-PAR ID:
10363122
Author(s) / Creator(s):
; ; ; ;
Publisher / Repository:
Springer Science + Business Media
Date Published:
Journal Name:
Journal of Biological Physics
Volume:
48
Issue:
1
ISSN:
0092-0606
Page Range / eLocation ID:
p. 37-53
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Schneidman-Duhovny, Dina (Ed.)
    We present OpenAWSEM and Open3SPN2, new cross-compatible implementations of coarse-grained models for protein (AWSEM) and DNA (3SPN2) molecular dynamics simulations within the OpenMM framework. These new implementations retain the chemical accuracy and intrinsic efficiency of the original models while adding GPU acceleration and the ease of forcefield modification provided by OpenMM’s Custom Forces software framework. By utilizing GPUs, we achieve around a 30-fold speedup in protein and protein-DNA simulations over the existing LAMMPS-based implementations running on a single CPU core. We showcase the benefits of OpenMM’s Custom Forces framework by devising and implementing two new potentials that allow us to address important aspects of protein folding and structure prediction and by testing the ability of the combined OpenAWSEM and Open3SPN2 to model protein-DNA binding. The first potential is used to describe the changes in effective interactions that occur as a protein becomes partially buried in a membrane. We also introduced an interaction to describe proteins with multiple disulfide bonds. Using simple pairwise disulfide bonding terms results in unphysical clustering of cysteine residues, posing a problem when simulating the folding of proteins with many cysteines. We now can computationally reproduce Anfinsen’s early Nobel prize winning experiments by using OpenMM’s Custom Forces framework to introduce a multi-body disulfide bonding term that prevents unphysical clustering. Our protein-DNA simulations show that the binding landscape is funneled towards structures that are quite similar to those found using experiments. In summary, this paper provides a simulation tool for the molecular biophysics community that is both easy to use and sufficiently efficient to simulate large proteins and large protein-DNA systems that are central to many cellular processes. These codes should facilitate the interplay between molecular simulations and cellular studies, which have been hampered by the large mismatch between the time and length scales accessible to molecular simulations and those relevant to cell biology. 
    more » « less
  2. Abstract

    Charged residues on the surface of proteins are critical for both protein stability and interactions. However, many proteins contain binding regions with a high net charge that may destabilize the protein but are useful for binding to oppositely charged targets. We hypothesized that these domains would be marginally stable, as electrostatic repulsion would compete with favorable hydrophobic collapse during folding. Furthermore, by increasing the salt concentration, we predict that these protein folds would be stabilized by mimicking some of the favorable electrostatic interactions that take place during target binding. We varied the salt and urea concentrations to probe the contributions of electrostatic and hydrophobic interactions for the folding of the yeast SH3 domain found in Abp1p. The SH3 domain was significantly stabilized with increased salt concentrations due to Debye–Huckel screening and a nonspecific territorial ion‐binding effect. Molecular dynamics and NMR show that sodium ions interact with all 15 acidic residues but do little to change backbone dynamics or overall structure. Folding kinetics experiments show that the addition of urea or salt primarily affects the folding rate, indicating that almost all the hydrophobic collapse and electrostatic repulsion occur in the transition state. After the transition state formation, modest yet favorable short‐range salt bridges are formed along with hydrogen bonds, as the native state fully folds. Thus, hydrophobic collapse offsets electrostatic repulsion to ensure this highly charged binding domain can still fold and be ready to bind to its charged peptide targets, a property that is likely evolutionarily conserved over 1 billion years.

     
    more » « less
  3. Abstract

    Heme is an essential and functionally versatile cofactor. Our understanding of how the environment of a heme in a protein tunes its function has benefited from spectroscopic and functional investigations of heme proteins and their variants with altered heme environments. Two properties of current interest are the conformation of the heme and hydrogen bonding to heme propionates. By combining nuclear magnetic resonance experiments and density functional theory calculations, both of these characteristics have been shown to influence the distribution of the singly occupied molecular orbital on the heme of ferricytochromec, which affects coupling to redox partners and electron‐transfer rates. In addition, heme conformation has been shown to tune reduction potential. These results reveal that subtle variations in heme conformation and in interactions with its propionates can have significant impacts on electron‐transfer activity.

     
    more » « less
  4. Cysteamine dioxygenase (ADO) has been reported to exhibit two distinct biological functions with a non-heme iron center. It catalyzes oxidation of both cysteamine in sulfur metabolism and N-terminal cysteine-containing proteins or peptides, such as regulator of G protein signaling 5 (RGS5). It thereby preserves oxygen homeostasis in a variety of physiological processes. However, little is known about its catalytic center and how it interacts with these two types of primary substrates in addition to O2. Here, using EPR, Mössbauer, and UV-Vis spectroscopies, we explored the binding mode of cysteamine and RGS5 to human and mouse ADO proteins in their physiologically relevant ferrous form. This characterization revealed that in the presence of nitric oxide as a spin probe and oxygen surrogate, both the small molecule and the peptide substrates coordinate to the iron center with their free thiols in a monodentate binding mode, in sharp contrast to binding behaviors observed in other thiol dioxygenases. We observed a substrate-bound B-type dinitrosyl iron center complex in ADO, suggesting the possibility of dioxygen binding to the iron ion in a side-on mode. Moreover, we observed a substrate-mediated reduction of the ferric to the ferrous oxidation state at the iron center. Subsequent MS analysis indicated corresponding disulfide formation of the substrates, suggesting that the presence of the substrate could reactivate ADO to defend against oxidative stress. The findings of this work contribute to the understanding of the substrate interaction in ADO and fill a gap in our knowledge of the substrate specificity of thiol dioxygenases. 
    more » « less
  5. Abstract

    Heme b (iron protoporphyrin IX) plays important roles in biology as a metallocofactor and signaling molecule. However, the targets of heme signaling and the network of proteins that mediate the exchange of heme from sites of synthesis or uptake to heme dependent or regulated proteins are poorly understood. Herein, we describe a quantitative mass spectrometry (MS)-based chemoproteomics strategy to identify exchange labile hemoproteins in human embryonic kidney HEK293 cells that may be relevant to heme signaling and trafficking. The strategy involves depleting endogenous heme with the heme biosynthetic inhibitor succinylacetone (SA), leaving putative heme-binding proteins in their apo-state, followed by the capture of those proteins using hemin–agarose resin, and finally elution and identification by MS. By identifying only those proteins that interact with high specificity to hemin–agarose relative to control beaded agarose in an SA-dependent manner, we have expanded the number of proteins and ontologies that may be involved in binding and buffering labile heme or are targets of heme signaling. Notably, these include proteins involved in chromatin remodeling, DNA damage response, RNA splicing, cytoskeletal organization, and vesicular trafficking, many of which have been associated with heme through complementary studies published recently. Taken together, these results provide support for the emerging role of heme in an expanded set of cellular processes from genome integrity to protein trafficking and beyond.

     
    more » « less