The conformational flexibility of the carboxy terminal residues 105-114 is a key modulator of the catalytic activity and stability of macrophage migration inhibitory factor

Farah El-Turk, Michele Cascella, Hajer Ouertatani-Sakouhi, Raghavendran Lakshmi Narayanan, Lin Leng, Richard Bucala, Markus Zweckstetter, Ursula Rothlisberger, Hilal A. Lashuel

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

Macrophage migration inhibitory factor (MIF) is a multifunctional protein and a major mediator of innate immunity. Although X-ray crystallography revealed that MIF exists as a homotrimer, its oligomerization state in vivo and the factors governing its oligomerization and stability remain poorly understood. The C-terminal region of MIF is highly conserved and participates in several intramolecular interactions that suggest a role in modulating the stability and biochemical activity of MIF. To determine the importance of these interactions, point mutations (A48P, L46A), insertions (P 107) at the monomer-monomer interfaces, and C-terminal deletion (Δ110-114NSTFA and Δ105-114NVGWNNSTFA) variants were designed and their structural properties, thermodynamic stability, oligomerization state, catalytic activity and receptor binding were characterized using a battery of biophysical methods. The C-terminal deletion mutants ΔC5 huMIF1-109 and ΔC10 huMIF1-104 were enzymatically inactive and thermodynamically less stable than wild type MIF. Analytical ultracentrifugation studies demonstrate that both C-terminal mutants sediment as trimers and exhibit similar binding to CD74 as the wild type protein. Disrupting the conformation of the C-terminal region 105-114 and increasing its conformational flexibility through the insertion of a proline residue at position 107 was sufficient to reproduce the structural, biochemical and thermodynamic properties of the deletion mutants. P107 MIF forms an enzymatically inactive trimer and exhibits reduced thermodynamic stability relative to the wild type protein. To provide a rationale for the changes induced by these mutations at the molecular level, we also performed molecular dynamics simulations on these mutants in comparison to the wild type MIF. Together, our studies demonstrate that intersubunit interactions involving the C-terminal region 105-114, including a salt-bridge interaction between Arg73 of one monomer and the carboxy terminus of a neighboring monomer, play critical roles in modulating tertiary structure stabilization, enzymatic activity, and thermodynamic stability of MIF, but not its oligomerization state and receptor binding properties. Our results suggest that targeting the C-terminal region could provide new strategies for allosteric modulation of MIF enzymatic activity and the development of novel inhibitors of MIF tautomerase activity.

Original languageEnglish
Pages (from-to)10740-10756
Number of pages17
JournalBiochemistry
Volume47
Issue number40
DOIs
Publication statusPublished - 7 Oct 2008
Externally publishedYes

Fingerprint

Macrophage Migration-Inhibitory Factors
Oligomerization
Thermodynamics
Modulators
Catalyst activity
Monomers
Thermodynamic stability
Proteins
X ray crystallography
Ultracentrifugation
X Ray Crystallography
Molecular Dynamics Simulation
Point Mutation
Innate Immunity
Proline
Conformations
Molecular dynamics
Structural properties
Sediments
Thermodynamic properties

ASJC Scopus subject areas

  • Biochemistry

Cite this

The conformational flexibility of the carboxy terminal residues 105-114 is a key modulator of the catalytic activity and stability of macrophage migration inhibitory factor. / El-Turk, Farah; Cascella, Michele; Ouertatani-Sakouhi, Hajer; Narayanan, Raghavendran Lakshmi; Leng, Lin; Bucala, Richard; Zweckstetter, Markus; Rothlisberger, Ursula; Lashuel, Hilal A.

In: Biochemistry, Vol. 47, No. 40, 07.10.2008, p. 10740-10756.

Research output: Contribution to journalArticle

El-Turk, F, Cascella, M, Ouertatani-Sakouhi, H, Narayanan, RL, Leng, L, Bucala, R, Zweckstetter, M, Rothlisberger, U & Lashuel, HA 2008, 'The conformational flexibility of the carboxy terminal residues 105-114 is a key modulator of the catalytic activity and stability of macrophage migration inhibitory factor', Biochemistry, vol. 47, no. 40, pp. 10740-10756. https://doi.org/10.1021/bi800603x
El-Turk, Farah ; Cascella, Michele ; Ouertatani-Sakouhi, Hajer ; Narayanan, Raghavendran Lakshmi ; Leng, Lin ; Bucala, Richard ; Zweckstetter, Markus ; Rothlisberger, Ursula ; Lashuel, Hilal A. / The conformational flexibility of the carboxy terminal residues 105-114 is a key modulator of the catalytic activity and stability of macrophage migration inhibitory factor. In: Biochemistry. 2008 ; Vol. 47, No. 40. pp. 10740-10756.
@article{f570b469bcdb4d19a7f7a5973f4fbf0b,
title = "The conformational flexibility of the carboxy terminal residues 105-114 is a key modulator of the catalytic activity and stability of macrophage migration inhibitory factor",
abstract = "Macrophage migration inhibitory factor (MIF) is a multifunctional protein and a major mediator of innate immunity. Although X-ray crystallography revealed that MIF exists as a homotrimer, its oligomerization state in vivo and the factors governing its oligomerization and stability remain poorly understood. The C-terminal region of MIF is highly conserved and participates in several intramolecular interactions that suggest a role in modulating the stability and biochemical activity of MIF. To determine the importance of these interactions, point mutations (A48P, L46A), insertions (P 107) at the monomer-monomer interfaces, and C-terminal deletion (Δ110-114NSTFA and Δ105-114NVGWNNSTFA) variants were designed and their structural properties, thermodynamic stability, oligomerization state, catalytic activity and receptor binding were characterized using a battery of biophysical methods. The C-terminal deletion mutants ΔC5 huMIF1-109 and ΔC10 huMIF1-104 were enzymatically inactive and thermodynamically less stable than wild type MIF. Analytical ultracentrifugation studies demonstrate that both C-terminal mutants sediment as trimers and exhibit similar binding to CD74 as the wild type protein. Disrupting the conformation of the C-terminal region 105-114 and increasing its conformational flexibility through the insertion of a proline residue at position 107 was sufficient to reproduce the structural, biochemical and thermodynamic properties of the deletion mutants. P107 MIF forms an enzymatically inactive trimer and exhibits reduced thermodynamic stability relative to the wild type protein. To provide a rationale for the changes induced by these mutations at the molecular level, we also performed molecular dynamics simulations on these mutants in comparison to the wild type MIF. Together, our studies demonstrate that intersubunit interactions involving the C-terminal region 105-114, including a salt-bridge interaction between Arg73 of one monomer and the carboxy terminus of a neighboring monomer, play critical roles in modulating tertiary structure stabilization, enzymatic activity, and thermodynamic stability of MIF, but not its oligomerization state and receptor binding properties. Our results suggest that targeting the C-terminal region could provide new strategies for allosteric modulation of MIF enzymatic activity and the development of novel inhibitors of MIF tautomerase activity.",
author = "Farah El-Turk and Michele Cascella and Hajer Ouertatani-Sakouhi and Narayanan, {Raghavendran Lakshmi} and Lin Leng and Richard Bucala and Markus Zweckstetter and Ursula Rothlisberger and Lashuel, {Hilal A.}",
year = "2008",
month = "10",
day = "7",
doi = "10.1021/bi800603x",
language = "English",
volume = "47",
pages = "10740--10756",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "40",

}

TY - JOUR

T1 - The conformational flexibility of the carboxy terminal residues 105-114 is a key modulator of the catalytic activity and stability of macrophage migration inhibitory factor

AU - El-Turk, Farah

AU - Cascella, Michele

AU - Ouertatani-Sakouhi, Hajer

AU - Narayanan, Raghavendran Lakshmi

AU - Leng, Lin

AU - Bucala, Richard

AU - Zweckstetter, Markus

AU - Rothlisberger, Ursula

AU - Lashuel, Hilal A.

PY - 2008/10/7

Y1 - 2008/10/7

N2 - Macrophage migration inhibitory factor (MIF) is a multifunctional protein and a major mediator of innate immunity. Although X-ray crystallography revealed that MIF exists as a homotrimer, its oligomerization state in vivo and the factors governing its oligomerization and stability remain poorly understood. The C-terminal region of MIF is highly conserved and participates in several intramolecular interactions that suggest a role in modulating the stability and biochemical activity of MIF. To determine the importance of these interactions, point mutations (A48P, L46A), insertions (P 107) at the monomer-monomer interfaces, and C-terminal deletion (Δ110-114NSTFA and Δ105-114NVGWNNSTFA) variants were designed and their structural properties, thermodynamic stability, oligomerization state, catalytic activity and receptor binding were characterized using a battery of biophysical methods. The C-terminal deletion mutants ΔC5 huMIF1-109 and ΔC10 huMIF1-104 were enzymatically inactive and thermodynamically less stable than wild type MIF. Analytical ultracentrifugation studies demonstrate that both C-terminal mutants sediment as trimers and exhibit similar binding to CD74 as the wild type protein. Disrupting the conformation of the C-terminal region 105-114 and increasing its conformational flexibility through the insertion of a proline residue at position 107 was sufficient to reproduce the structural, biochemical and thermodynamic properties of the deletion mutants. P107 MIF forms an enzymatically inactive trimer and exhibits reduced thermodynamic stability relative to the wild type protein. To provide a rationale for the changes induced by these mutations at the molecular level, we also performed molecular dynamics simulations on these mutants in comparison to the wild type MIF. Together, our studies demonstrate that intersubunit interactions involving the C-terminal region 105-114, including a salt-bridge interaction between Arg73 of one monomer and the carboxy terminus of a neighboring monomer, play critical roles in modulating tertiary structure stabilization, enzymatic activity, and thermodynamic stability of MIF, but not its oligomerization state and receptor binding properties. Our results suggest that targeting the C-terminal region could provide new strategies for allosteric modulation of MIF enzymatic activity and the development of novel inhibitors of MIF tautomerase activity.

AB - Macrophage migration inhibitory factor (MIF) is a multifunctional protein and a major mediator of innate immunity. Although X-ray crystallography revealed that MIF exists as a homotrimer, its oligomerization state in vivo and the factors governing its oligomerization and stability remain poorly understood. The C-terminal region of MIF is highly conserved and participates in several intramolecular interactions that suggest a role in modulating the stability and biochemical activity of MIF. To determine the importance of these interactions, point mutations (A48P, L46A), insertions (P 107) at the monomer-monomer interfaces, and C-terminal deletion (Δ110-114NSTFA and Δ105-114NVGWNNSTFA) variants were designed and their structural properties, thermodynamic stability, oligomerization state, catalytic activity and receptor binding were characterized using a battery of biophysical methods. The C-terminal deletion mutants ΔC5 huMIF1-109 and ΔC10 huMIF1-104 were enzymatically inactive and thermodynamically less stable than wild type MIF. Analytical ultracentrifugation studies demonstrate that both C-terminal mutants sediment as trimers and exhibit similar binding to CD74 as the wild type protein. Disrupting the conformation of the C-terminal region 105-114 and increasing its conformational flexibility through the insertion of a proline residue at position 107 was sufficient to reproduce the structural, biochemical and thermodynamic properties of the deletion mutants. P107 MIF forms an enzymatically inactive trimer and exhibits reduced thermodynamic stability relative to the wild type protein. To provide a rationale for the changes induced by these mutations at the molecular level, we also performed molecular dynamics simulations on these mutants in comparison to the wild type MIF. Together, our studies demonstrate that intersubunit interactions involving the C-terminal region 105-114, including a salt-bridge interaction between Arg73 of one monomer and the carboxy terminus of a neighboring monomer, play critical roles in modulating tertiary structure stabilization, enzymatic activity, and thermodynamic stability of MIF, but not its oligomerization state and receptor binding properties. Our results suggest that targeting the C-terminal region could provide new strategies for allosteric modulation of MIF enzymatic activity and the development of novel inhibitors of MIF tautomerase activity.

UR - http://www.scopus.com/inward/record.url?scp=53249090254&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=53249090254&partnerID=8YFLogxK

U2 - 10.1021/bi800603x

DO - 10.1021/bi800603x

M3 - Article

VL - 47

SP - 10740

EP - 10756

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 40

ER -