Article
A Water-Bridged Cysteine-Cysteine Redox
Regulation Mechanism in Bacterial
Protein Tyrosine Phosphatases
Jean B. Bertoldo,1,2 Tiago Rodrigues,3,8 Lavinia Dunsmore,1,8 Francesco A. Aprile,1,8
Marta C. Marques,3,8 Leonardo A. Rosado,2 Omar Boutureira,1 Thomas B. Steinbrecher,4
Woody Sherman,5,7 Francisco Corzana,6 Herna´n Terenzi,2 and Gonc¸alo J.L. Bernardes1,3,9,
*
SUMMARY
The Bigger Picture
The emergence of
The emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains
highlights the need to develop more efficacious and potent drugs. However,
this goal is dependent on a comprehensive understanding of Mtb virulence pro-
tein effectors at the molecular level. Here, we used a post-expression cysteine
(Cys)-to-dehydrolanine (Dha) chemical editing strategy to identify a water-medi-
ated motif that modulates accessibility of the protein tyrosine phosphatase
A (PtpA) catalytic pocket. Importantly, this water-mediated Cys-Cys non-cova-
lent motif is also present in the phosphatase SptpA from Staphylococcus aureus,
which suggests a potentially preserved structural feature among bacterial tyro-
sine phosphatases. The identification of this structural water provides insight
into the known resistance of Mtb PtpA to the oxidative conditions that prevail
within an infected host macrophage. This strategy could be applied to extend
the understanding of the dynamics and function(s) of proteins in their native
state and ultimately aid in the design of small-molecule modulators.
Mycobacterium tuberculosis
(Mtb) resistance is a serious threat
to public health. However, the
quest for more efficient drugs
against Mtb is hampered by the
lack of a detailed understanding
of Mtb virulence protein effectors.
Here, we describe the swift
modification of select Cys
residues in multi-Cys proteins
directly through chemistry. New
insights into the biochemistry of
emerging bacterial drug targets
were obtained. We reveal a water
Cys-Cys bridging mechanism that
offers an explanation for the
known resistance of Mtb protein
tyrosine phosphatase A (PtpA) to
the oxidative conditions that
prevail within an infected host
macrophage. This water Cys-Cys
bridge motif is also found in the
phosphatase SptpA from
INTRODUCTION
Tuberculosis affects millions of people each year and is a leading cause of deaths world-
wide.1 The emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains
is linked to the ability of Mtb to overcome host defenses, especially macrophage diges-
tion and overoxidation,2,3 pressuring the long-standing endeavor of disease eradica-
tion.4 Once inside the macrophage vacuole, Mtb circumvents the proteolysis machinery
by inhibiting phagosome maturation and its fusion with lysosome.5 Among others, pro-
tein tyrosine phosphatase A (PtpA) is a key player for Mtb survival in this oxidative envi-
ronment. PtpA is secreted into the macrophage cytosol and interferes directly with
phagosome maturation by disrupting key components of the macrophage endocytic
pathway.6,7 However, as macrophages produce reactive oxygen and nitrogen species
as a defensemechanism against Mtb,8 proteins, including PtpA, arelikelyto be inhibited
under oxidative conditions. Protein tyrosine phosphatases (PTPs) contain multiple Cys
residues that play a paramount role regulating signaling pathways (Figure 1A).9–11 The
formation of a disulfide bridge between the catalytic Cys and a backdoor Cys residue
located within the catalytic pocket is a structural feature that can finely control the redox
mechanism of PTPs.12 However, such regulating mechanism(s) that delay oxidative
inactivation remain elusive for PtpA.
Staphylococcus aureus,
suggesting its potential
conserved structural role. The
rationalization of the unique
features of PtpA, an important
target for Mtb drug discovery,
could now be used in the design
of novel small-molecule
modulators.
Typically, the relationship between amino acid sequence and protein activity and func-
tion is determined through site-directed mutagenesis.13 However, this technique is
Chem 3, 665–677, October 12, 2017 ª 2017 The Authors. Published by Elsevier Inc. 665