Proton Bridging in Catalysis by and Inhibition of Serine Proteases of the Blood Cascade System

Inquiries into the participation of short hydrogen bonds in stabilizing transition states and intermediate states in the thrombin, factor Xa, plasmin and activated protein C–catalyzed reactions revealed that specific binding of effectors at Sn, n = 1–4 and S’n, n = 1–3 and at remote exosites elicit complex patterns of hydrogen bonding and involve water networks. The methods employed that yielded these discoveries include; (1) kinetics, especially partial or full kinetic deuterium solvent isotope effects with short cognate substrates and also with the natural substrates, (2) kinetic and structural probes, particularly low-field high-resolution nuclear magnetic resonance (1H NMR), of mechanism-based inhibitors and substrate-mimic peptide inhibitors. Short hydrogen bonds form at the transition states of the catalytic reactions at the active site of the enzymes as they do with mechanism-based covalent inhibitors of thrombin. The emergence of short hydrogen bonds at the binding interface of effectors and thrombin at remote exosites has recently gained recognition. Herein, I describe our contribution, a confirmation of this discovery, by low-field 1H NMR. The principal conclusion of this review is that proton sharing at distances below the sum of van der Waals radii of the hydrogen and both donor and acceptor atoms contribute to the remarkable catalytic prowess of serine proteases of the blood clotting system and other enzymes that employ acid-base catalysis. Proton bridges also play a role in tight binding in proteins and at exosites, i.e., allosteric sites, of enzymes.

Download Full-text

Short hydrogen bonds in the catalytic mechanism of serine proteases

Journal of the Serbian Chemical Society ◽

10.2298/jsc0804393l ◽

2008 ◽

Vol 73 (4) ◽

pp. 393-403 ◽

Cited By ~ 5

Author(s):

Vladimir Leskovac ◽

Svetlana Trivic ◽

Draginja Pericin ◽

Mira Popovic ◽

Julijan Kandrac

Keyword(s):

Hydrogen Bond ◽

Hydrogen Bonds ◽

Active Site ◽

Serine Proteases ◽

Active Sites ◽

Catalytic Mechanism ◽

Data Bank ◽

Ground State Structure ◽

Low Barrier Hydrogen Bonds ◽

Short Hydrogen Bonds

The survey of crystallographic data from the Protein Data Bank for 37 structures of trypsin and other serine proteases at a resolution of 0.78-1.28 ? revealed the presence of hydrogen bonds in the active site of the enzymes, which are formed between the catalytic histidine and aspartate residues and are on average 2.7 ? long. This is the typical bond length for normal hydrogen bonds. The geometric properties of the hydrogen bonds in the active site indicate that the H atom is not centered between the heteroatoms of the catalytic histidine and aspartate residues in the active site. Taken together, these findings exclude the possibility that short "low-barrier" hydrogen bonds are formed in the ground state structure of the active sites examined in this work. Some time ago, it was suggested by Cleland that the "low-barrier hydrogen bond" hypothesis is operative in the catalytic mechanism of serine proteases, and requires the presence of short hydrogen bonds around 2.4 ? long in the active site, with the H atom centered between the catalytic heteroatoms. The conclusions drawn from this work do not exclude the validity of the "low-barrier hydrogen bond" hypothesis at all, but they merely do not support it in this particular case, with this particular class of enzymes.

Download Full-text

Symmetry and 1H NMR chemical shifts of short hydrogen bonds: impact of electronic and nuclear quantum effects

Physical Chemistry Chemical Physics ◽

10.1039/c9cp06840f ◽

2020 ◽

Vol 22 (9) ◽

pp. 4884-4895 ◽

Cited By ~ 3

Author(s):

Shengmin Zhou ◽

Lu Wang

Keyword(s):

Hydrogen Bonds ◽

1H Nmr ◽

Chemical Shifts ◽

Quantum Effects ◽

Nmr Chemical Shifts ◽

H Nmr ◽

Nuclear Quantum Effects ◽

Short Hydrogen Bonds

Electronic and nuclear quantum effects determine the symmetry and highly downfield 1H NMR chemical shifts of short hydrogen bonds.

Download Full-text

Symmetry and 1H NMR chemical shifts of short hydrogen bonds

10.1021/scimeetings.0c00636 ◽

2020 ◽

Author(s):

Lu Wang

Keyword(s):

Hydrogen Bonds ◽

1H Nmr ◽

Chemical Shifts ◽

Nmr Chemical Shifts ◽

Short Hydrogen Bonds

Download Full-text

Effective prediction of short hydrogen bonds in proteins via machine learning method

Scientific Reports ◽

10.1038/s41598-021-04306-4 ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Shengmin Zhou ◽

Yuanhao Liu ◽

Sijian Wang ◽

Lu Wang

Keyword(s):

Machine Learning ◽

Hydrogen Bond ◽

Amino Acid ◽

Hydrogen Bonds ◽

Protein Structures ◽

Decomposition Analysis ◽

Energy Decomposition Analysis ◽

Donor And Acceptor ◽

Wide Range ◽

Short Hydrogen Bonds

AbstractShort hydrogen bonds (SHBs), whose donor and acceptor heteroatoms lie within 2.7 Å, exhibit prominent quantum mechanical characters and are connected to a wide range of essential biomolecular processes. However, exact determination of the geometry and functional roles of SHBs requires a protein to be at atomic resolution. In this work, we analyze 1260 high-resolution peptide and protein structures from the Protein Data Bank and develop a boosting based machine learning model to predict the formation of SHBs between amino acids. This model, which we name as machine learning assisted prediction of short hydrogen bonds (MAPSHB), takes into account 21 structural, chemical and sequence features and their interaction effects and effectively categorizes each hydrogen bond in a protein to a short or normal hydrogen bond. The MAPSHB model reveals that the type of the donor amino acid plays a major role in determining the class of a hydrogen bond and that the side chain Tyr-Asp pair demonstrates a significant probability of forming a SHB. Combining electronic structure calculations and energy decomposition analysis, we elucidate how the interplay of competing intermolecular interactions stabilizes the Tyr-Asp SHBs more than other commonly observed combinations of amino acid side chains. The MAPSHB model, which is freely available on our web server, allows one to accurately and efficiently predict the presence of SHBs given a protein structure with moderate or low resolution and will facilitate the experimental and computational refinement of protein structures.

Download Full-text

Unusual Spectroscopic and Electric Field Sensitivity of a Chromophore with Short Hydrogen Bond: GFP and PYP as Model Systems

10.26434/chemrxiv.12847913.v1 ◽

2020 ◽

Author(s):

Chi-Yun Lin ◽

Steven Boxer

Keyword(s):

Hydrogen Bond ◽

Hydrogen Bonds ◽

Electric Field ◽

External Electric Field ◽

Heavy Atom ◽

Model Systems ◽

Donor And Acceptor ◽

Short Hydrogen Bond ◽

Structural Methods ◽

Short Hydrogen Bonds

<p> Short hydrogen bonds, with heavy-atom distances less than 2.7 Å, are believed to exhibit proton delocalization and their possible role in catalysis has been widely debated. While spectroscopic and/or structural methods are usually employed to study the degree of proton delocalization, ambiguities still arise and no direct information on the corresponding potential energy surface is obtained. Here we apply an external electric field to perturb the short hydrogen bond(s) within a collection of green fluorescent protein S65T/H148D variants and photoactive yellow protein mutants, where the chromophore participates in the short hydrogen bond(s) and serves as an optical probe of the proton position. As the proton is charged, its position may shift in response to the external electric field, and the chromophore’s electronic absorption can thus reflect the ease of proton transfer. The results suggest that low-barrier hydrogen bonds are not present within these proteins even when proton affinities between donor and acceptor are closely matched. Exploiting the chromophores as pre-calibrated electrostatic probes, the covalency of short hydrogen bonds as a non-electrostatic component was also revealed. No clear evidence was found for a possible contribution of unusually large polarizabilities of short hydrogen bonds due to proton delocalization; a theoretical framework for this interesting phenomenon is developed.<br></p>

Download Full-text

Unusual Spectroscopic and Electric Field Sensitivity of a Chromophore with Short Hydrogen Bond: GFP and PYP as Model Systems

10.26434/chemrxiv.12847913 ◽

2020 ◽

Author(s):

Chi-Yun Lin ◽

Steven Boxer

Keyword(s):

Hydrogen Bond ◽

Hydrogen Bonds ◽

Electric Field ◽

External Electric Field ◽

Heavy Atom ◽

Model Systems ◽

Donor And Acceptor ◽

Short Hydrogen Bond ◽

Structural Methods ◽

Short Hydrogen Bonds

Download Full-text

Sensitivity of Intra- and Intermolecular Interactions of Benzo[h]quinoline from Car–Parrinello Molecular Dynamics and Electronic Structure Inspection

International Journal of Molecular Sciences ◽

10.3390/ijms22105220 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5220

Author(s):

Jarosław J. Panek ◽

Joanna Zasada ◽

Bartłomiej M. Szyja ◽

Beata Kizior ◽

Aneta Jezierska

Keyword(s):

Molecular Dynamics ◽

Electronic Structure ◽

Hydrogen Bonds ◽

Density Functional ◽

Quantum Effects ◽

Potential Of Mean Force ◽

Probability Density Distribution ◽

Donor And Acceptor ◽

Nuclear Quantum Effects ◽

Vibrational Features

The O-H...N and O-H...O hydrogen bonds were investigated in 10-hydroxybenzo[h]quinoline (HBQ) and benzo[h]quinoline-2-methylresorcinol complex in vacuo, solvent and crystalline phases. The chosen systems contain analogous donor and acceptor moieties but differently coupled (intra- versus intermolecularly). Car–Parrinello molecular dynamics (CPMD) was employed to shed light onto principle components of interactions responsible for the self-assembly. It was applied to study the dynamics of the hydrogen bonds and vibrational features as well as to provide initial geometries for incorporation of quantum effects and electronic structure studies. The vibrational features were revealed using Fourier transformation of the autocorrelation function of atomic velocity and by inclusion of nuclear quantum effects on the O-H stretching solving vibrational Schrödinger equation a posteriori. The potential of mean force (Pmf) was computed for the whole trajectory to derive the probability density distribution and for the O-H stretching mode from the proton vibrational eigenfunctions and eigenvalues incorporating statistical sampling and nuclear quantum effects. The electronic structure changes of the benzo[h]quinoline-2-methylresorcinol dimer and trimers were studied based on Constrained Density Functional Theory (CDFT) whereas the Electron Localization Function (ELF) method was applied for all systems. It was found that the bridged proton is localized on the donor side in both investigated systems in vacuo. The crystalline phase simulations indicated bridged proton-sharing and transfer events in HBQ. These effects are even more pronounced when nuclear quantization is taken into account, and the quantized Pmf allows the proton to sample the acceptor area more efficiently. The CDFT indicated the charge depletion at the bridged proton for the analyzed dimer and trimers in solvent. The ELF analysis showed the presence of the isolated proton (a signature of the strongest hydrogen bonds) only in some parts of the HBQ crystal simulation. The collected data underline the importance of the intramolecular coupling between the donor and acceptor moieties.

Download Full-text

Identification of the SV2 protein receptor-binding site of botulinum neurotoxin type E

Biochemical Journal ◽

10.1042/bj20130391 ◽

2013 ◽

Vol 453 (1) ◽

pp. 37-47 ◽

Cited By ~ 31

Author(s):

Stefan Mahrhold ◽

Jasmin Strotmeier ◽

Consuelo Garcia-Rodriguez ◽

Jianlong Lou ◽

James D. Marks ◽

...

Keyword(s):

Binding Site ◽

Synaptic Vesicle ◽

Specific Binding ◽

Cell Surface Receptor ◽

Vesicle Membrane ◽

Receptor Binding Site ◽

Binding Interface ◽

Protein Receptor ◽

Surface Receptor ◽

Extended Surface

The highly specific binding and uptake of BoNTs (botulinum neurotoxins; A–G) into peripheral cholinergic motoneurons turns them into the most poisonous substances known. Interaction with gangliosides accumulates the neurotoxins on the plasma membrane and binding to a synaptic vesicle membrane protein leads to neurotoxin endocytosis. SV2 (synaptic vesicle glycoprotein 2) mediates the uptake of BoNT/A and /E, whereas Syt (synaptotagmin) is responsible for the endocytosis of BoNT/B and /G. The Syt-binding site of the former was identified by co-crystallization and mutational analyses. In the present study we report the identification of the SV2-binding interface of BoNT/E. Mutations interfering with SV2 binding were located at a site that corresponds to the Syt-binding site of BoNT/B and at an extended surface area located on the back of the conserved ganglioside-binding site, comprising the N- and C-terminal half of the BoNT/E-binding domain. Mutations impairing the affinity also reduced the neurotoxicity of full-length BoNT/E at mouse phrenic nerve hemidiaphragm preparations demonstrating the crucial role of the identified binding interface. Furthermore, we show that a monoclonal antibody neutralizes BoNT/E activity because it directly interferes with the BoNT/E–SV2 interaction. The results of the present study suggest a novel mode of binding for BoNTs that exploit SV2 as a cell surface receptor.

Download Full-text