A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

Summary

Lanthipeptide synthetases catalyze multistep reactions during the biosynthesis of peptide natural products. Here, we describe a continuous, bottom-up, hydrogen-deuterium exchange mass spectrometry (HDX-MS) workflow that can be employed to study the conformational dynamics of lanthipeptide synthetases, as well as other similar enzymes involved in peptide natural product biosynthesis.

Abstract

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and enzyme-substrate interactions. Among its many benefits, HDX-MS consumes only small amounts of material, can be performed under near native conditions without the need for enzyme/substrate labeling, and can provide spatially resolved information on enzyme conformational dynamics−even for large enzymes and multiprotein complexes. The method is initiated by the dilution of the enzyme of interest into buffer prepared in D₂O. This triggers the exchange of protium in peptide bond amides (N-H) with deuterium (N-D). At the desired exchange time points, reaction aliquots are quenched, the enzyme is proteolyzed into peptides, the peptides are separated by ultra-performance liquid chromatography (UPLC), and the change in mass of each peptide (due to the exchange of hydrogen for deuterium) is recorded by MS. The amount of deuterium uptake by each peptide is strongly dependent on the local hydrogen bonding environment of that peptide. Peptides present in very dynamic regions of the enzyme exchange deuterium very rapidly, whereas peptides derived from well-ordered regions undergo exchange much more slowly. In this manner, the HDX rate reports on local enzyme conformational dynamics. Perturbations to deuterium uptake levels in the presence of different ligands can then be used to map ligand binding sites, identify allosteric networks, and to understand the role of conformational dynamics in enzyme function. Here, we illustrate how we have used HDX-MS to better understand the biosynthesis of a type of peptide natural products called lanthipeptides. Lanthipeptides are genetically encoded peptides that are post-translationally modified by large, multifunctional, conformationally dynamic enzymes that are difficult to study with traditional structural biology approaches. HDX-MS provides an ideal and adaptable platform for investigating the mechanistic properties of these types of enzymes.

Introduction

Proteins are structurally dynamic molecules that sample different conformations on time scales ranging from femtosecond-scale bond vibration to rearrangements of entire protein domains which can occur over many seconds¹. These conformational fluctuations are often critical aspects of enzyme/protein function. For example, conformational changes induced by ligand binding are often critically important for modulating enzyme function, either by organizing active site residues needed for catalysis, defining substrate binding sites in sequential kinetic mechanisms, shielding reactive intermediates from the environment, or by modulating enzyme functio....

Protocol

1. Preparation of deuterated reagents and enzyme stock solutions

1. Prepare reagents needed for the HDX reactions (including any buffers, salts, substrates, ligands, etc.) as 100−200x concentrated stock solutions in D₂O (99.9% atom fraction D). Prepare at least 50 mL of buffer stock solution.
   NOTE: For characterization of HalM2, the following solutions were prepared: 500 mM MgCl₂, 100 mM tris(2-carboxyethyl)phosphine (TCEP), 750 mM ATP (in HEPES buffer), 800 mM HEPES pD 7.1, 500 µM HalA2, and 500 mM AMPPNP.
2. Freeze and lyophilize the stock solutions to dryness.
3. Re-dissolve in D₂O, and repeat lyophilizat....

Results

It is necessary to assess the quality of the proteolytic digestion and the reproducibility of the workflow for each set of sample injections. Thus, prior to performing HDX-MS assays, it is essential to establish effective conditions for the proteolysis of the protein of interest, for the separation of peptides using reverse phase liquid chromatography and gas phase ion mobility, and for the detection of peptides using MS. For this purpose, the reference samples for the protein of interest (collected in the absence of deu.......

Discussion

The HDX-MS workflow presented in this protocol provides a remarkably robust platform for mapping the spatial distribution of structurally dynamic elements in proteins and for investigating how these dynamics change in response to perturbation (ligand binding, enzyme mutagenesis, etc.). HDX-MS holds several distinct advantages over other structural biology approaches that are commonly used to investigate conformational dynamics. Most notably, only small quantities of protein are needed. Using the workflow described herein.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
[glu-1]-fibrinopeptide B (Glu-Fib)	BioBasic	NA
0.5 mL Amicon Ultracel 10k centrifugal filtration device (Millipore)	Milipore Sigma	UFC501096
acetonitrile	Fisher	A955-1
AMP-PNP	SIGMA	A2647-25MG
ATP	SIGMA	a2383-5G
D2O	ALDRICH	435767-100G
formic acid	Thermo Fisher	28905
guanidine-HCl	VWR	97063-764
HEPES	Fisher	BP310-1
Magnesium chloride	SiGMA-Aldrich	63068-250G
Potassium chloride	BioBasic	PB0440
potassium phosphate	BioBasic	PB0445
TCEP Hydrochloride	TRC Canada	T012500	peptide was synthesized upon request
Name of Material/ Equipment	Company	Catalog Number	Comments/Description
software
Deuteros	Andy M C Lau, et al	version 1.08
DynamX	Waters	version 3.0
MassLynx	Waters	version 4.1
Protein Lynx Global Server (PLGS)	Waters	version 3.0.3
PyMOL	Schrödinger	version 2.2.2
Name of Material/ Equipment	Company	Catalog Number	Comments/Description
Instrument and equipment
ACQUITY UPLC BEH C18 analytical Column	Waters	186002346
ACQUITY UPLC BEH C8 VanGuard Pre-column	Waters	186003978
ACQUITY UPLC M-Class HDX System	Waters
HDX Manager	Waters
microtip pH electrode	Thermo Fisher	13-620-291
Waters Enzymate BEH column or Pepsin solumn	Waters	186007233
Waters Synapt G2-Si	Waters