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Structural and functional characterization of a cold‐adapted stand‐alone TPM domain reveals a relationship between dynamics and phosphatase activity
Author(s) -
Pellizza Leonardo A.,
Smal Clara,
Ithuralde Raúl E.,
Turjanski Adrián G.,
Cicero Daniel O.,
Arán Martín
Publication year - 2016
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13929
Subject(s) - hydrolase , molecular dynamics , chemistry , biophysics , function (biology) , phosphatase , enzyme , divalent , protein domain , crystallography , biochemistry , biology , microbiology and biotechnology , computational chemistry , organic chemistry , gene
The TPM domain constitutes a family of recently characterized protein domains that are present in most living organisms. Although some progress has been made in understanding the cellular role of TPM ‐containing proteins, the relationship between structure and function is not clear yet. We have recently solved the solution and crystal structure of one TPM domain ( BA 42) from the Antarctic bacterium Bizionia argentinensis . In this work, we demonstrate that BA 42 has phosphoric‐monoester hydrolase activity. The activity of BA 42 is strictly dependent on the binding of divalent metals and retains nearly 70% of the maximum at 4 °C, a typical characteristic of cold‐adapted enzymes. From HSQC , 15 N relaxation measurements, and molecular dynamics studies, we determine that the flexibility of the crossing loops was associated to the protein activity. Thermal unfolding experiments showed that the local increment in flexibility of Mg 2+ ‐bound BA 42, when compared with Ca 2+ ‐bound BA 42, is associated to a decrease in global protein stability. Finally, through mutagenesis experiments, we unambiguously demonstrate that the region comprising the metal‐binding site participates in the catalytic mechanism. The results shown here contribute to the understanding of the relationship between structure and function of this new family of TPM domains providing important cues on the regulatory role of Mg 2+ and Ca 2+ and the molecular mechanism underlying enzyme activity at low temperatures.