Intra- and inter-molecular effects of a conserved arginine residue of neuronal and inducible nitric oxide synthases on FMN and calmodulin binding

Satya Prakash Panda; Srikanth R. Polusani; Dean L. Kellogg; Priya Venkatakrishnan; Madeline G. Roman; Borries Demeler; Bettie Sue S. Masters; Linda J. Roman

doi:10.1016/j.abb.2013.03.004

Intra- and inter-molecular effects of a conserved arginine residue of neuronal and inducible nitric oxide synthases on FMN and calmodulin binding

Satya Prakash Panda
, Srikanth R. Polusani
, Dean L. Kellogg
, Priya Venkatakrishnan
, Madeline G. Roman
, Borries Demeler
, Bettie Sue S. Masters
, Linda J. Roman

Chemistry and Biochemistry

University of Texas Health Science Center at San Antonio

Research output: Contribution to journal › Article › peer-review

2 Scopus citations

Abstract

Nitric oxide synthases (NOSs) synthesize nitric oxide (NO), a signaling molecule, from l-arginine, utilizing electrons from NADPH. NOSs are flavo-hemo proteins, with two flavin molecules (FAD and FMN) and one heme per monomer, which require the binding of calcium/calmodulin (Ca²⁺/CaM) to produce NO. It is therefore important to understand the molecular factors influencing CaM binding from a structure/function perspective. A crystal structure of the CaM-bound iNOS FMN-binding domain predicted a salt bridge between R536 of human iNOS and E47 of CaM. To characterize the interaction between the homologous Arg of rat nNOS (R753) and murine iNOS (R530) with CaM, the Arg was mutated to Ala and, in iNOS, to Glu. The mutation weakens the interaction between nNOS and CaM, decreasing affinity by ∼3-fold. The rate of electron transfer from FMN is greatly attenuated; however, little effect on electron transfer from FAD is observed. The mutated proteins showed reduced FMN binding, from 20% to 60%, suggesting an influence of this residue on FMN incorporation. The weakened FMN binding may be due to conformational changes caused by the arginine mutation. Our data show that this Arg residue plays an important role in CaM binding and influences FMN binding.

Original language	English
Pages (from-to)	88-94
Number of pages	7
Journal	Archives of Biochemistry and Biophysics
Volume	533
Issue number	1-2
DOIs	https://doi.org/10.1016/j.abb.2013.03.004
State	Published - May 2013

Funding

The authors wish to thank Dr. Jung-Ja Kim, Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI-53226 for her critical reading of the manuscript and suggestions. Supported by NIH GM052419 to BSSM and LJR. BSSM is the Robert A. Welch Distinguished Professor in Chemistry (AQ-0012). The development of the UltraScan software is supported by the National Institutes of Health through grant RR022200 (to BD). Supercomputer time allocations were provided through National Science Foundation grant TG-MCB070038 (to BD). We acknowledge the support of the San Antonio Cancer Institute grant P30 CA054174 for the Center for Analytical Ultracentrifugation of Macromolecular Assemblies at the University of Texas Health Science Center at San Antonio.

Funders	Funder number
P30 CA054174
TG-MCB070038
RR022200
R01GM052419
University of Texas Health Science Center at San Antonio

Keywords

Calmodulin
Electron transfer
Flavoprotein
Nitric oxide synthase
Reductase

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10.1016/j.abb.2013.03.004

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Panda, S. P., Polusani, S. R., Kellogg, D. L., Venkatakrishnan, P., Roman, M. G., Demeler, B., Masters, B. S. S., & Roman, L. J. (2013). Intra- and inter-molecular effects of a conserved arginine residue of neuronal and inducible nitric oxide synthases on FMN and calmodulin binding. Archives of Biochemistry and Biophysics, 533(1-2), 88-94. https://doi.org/10.1016/j.abb.2013.03.004

@article{10882b78c45e41ebb33e2b60c5ecce36,

title = "Intra- and inter-molecular effects of a conserved arginine residue of neuronal and inducible nitric oxide synthases on FMN and calmodulin binding",

abstract = "Nitric oxide synthases (NOSs) synthesize nitric oxide (NO), a signaling molecule, from l-arginine, utilizing electrons from NADPH. NOSs are flavo-hemo proteins, with two flavin molecules (FAD and FMN) and one heme per monomer, which require the binding of calcium/calmodulin (Ca2+/CaM) to produce NO. It is therefore important to understand the molecular factors influencing CaM binding from a structure/function perspective. A crystal structure of the CaM-bound iNOS FMN-binding domain predicted a salt bridge between R536 of human iNOS and E47 of CaM. To characterize the interaction between the homologous Arg of rat nNOS (R753) and murine iNOS (R530) with CaM, the Arg was mutated to Ala and, in iNOS, to Glu. The mutation weakens the interaction between nNOS and CaM, decreasing affinity by ∼3-fold. The rate of electron transfer from FMN is greatly attenuated; however, little effect on electron transfer from FAD is observed. The mutated proteins showed reduced FMN binding, from 20\% to 60\%, suggesting an influence of this residue on FMN incorporation. The weakened FMN binding may be due to conformational changes caused by the arginine mutation. Our data show that this Arg residue plays an important role in CaM binding and influences FMN binding.",

keywords = "Calmodulin, Electron transfer, Flavoprotein, Nitric oxide synthase, Reductase",

author = "Panda, \{Satya Prakash\} and Polusani, \{Srikanth R.\} and Kellogg, \{Dean L.\} and Priya Venkatakrishnan and Roman, \{Madeline G.\} and Borries Demeler and Masters, \{Bettie Sue S.\} and Roman, \{Linda J.\}",

note = "Funding Information: The authors wish to thank Dr. Jung-Ja Kim, Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI-53226 for her critical reading of the manuscript and suggestions. Supported by NIH GM052419 to BSSM and LJR. BSSM is the Robert A. Welch Distinguished Professor in Chemistry (AQ-0012). The development of the UltraScan software is supported by the National Institutes of Health through grant RR022200 (to BD). Supercomputer time allocations were provided through National Science Foundation grant TG-MCB070038 (to BD). We acknowledge the support of the San Antonio Cancer Institute grant P30 CA054174 for the Center for Analytical Ultracentrifugation of Macromolecular Assemblies at the University of Texas Health Science Center at San Antonio. ",

year = "2013",

month = may,

doi = "10.1016/j.abb.2013.03.004",

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pages = "88--94",

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issn = "0003-9861",

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Panda, SP, Polusani, SR, Kellogg, DL, Venkatakrishnan, P, Roman, MG, Demeler, B, Masters, BSS & Roman, LJ 2013, 'Intra- and inter-molecular effects of a conserved arginine residue of neuronal and inducible nitric oxide synthases on FMN and calmodulin binding', Archives of Biochemistry and Biophysics, vol. 533, no. 1-2, pp. 88-94. https://doi.org/10.1016/j.abb.2013.03.004

TY - JOUR

T1 - Intra- and inter-molecular effects of a conserved arginine residue of neuronal and inducible nitric oxide synthases on FMN and calmodulin binding

AU - Panda, Satya Prakash

AU - Polusani, Srikanth R.

AU - Kellogg, Dean L.

AU - Venkatakrishnan, Priya

AU - Roman, Madeline G.

AU - Demeler, Borries

AU - Masters, Bettie Sue S.

AU - Roman, Linda J.

N1 - Funding Information: The authors wish to thank Dr. Jung-Ja Kim, Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI-53226 for her critical reading of the manuscript and suggestions. Supported by NIH GM052419 to BSSM and LJR. BSSM is the Robert A. Welch Distinguished Professor in Chemistry (AQ-0012). The development of the UltraScan software is supported by the National Institutes of Health through grant RR022200 (to BD). Supercomputer time allocations were provided through National Science Foundation grant TG-MCB070038 (to BD). We acknowledge the support of the San Antonio Cancer Institute grant P30 CA054174 for the Center for Analytical Ultracentrifugation of Macromolecular Assemblies at the University of Texas Health Science Center at San Antonio.

PY - 2013/5

Y1 - 2013/5

N2 - Nitric oxide synthases (NOSs) synthesize nitric oxide (NO), a signaling molecule, from l-arginine, utilizing electrons from NADPH. NOSs are flavo-hemo proteins, with two flavin molecules (FAD and FMN) and one heme per monomer, which require the binding of calcium/calmodulin (Ca2+/CaM) to produce NO. It is therefore important to understand the molecular factors influencing CaM binding from a structure/function perspective. A crystal structure of the CaM-bound iNOS FMN-binding domain predicted a salt bridge between R536 of human iNOS and E47 of CaM. To characterize the interaction between the homologous Arg of rat nNOS (R753) and murine iNOS (R530) with CaM, the Arg was mutated to Ala and, in iNOS, to Glu. The mutation weakens the interaction between nNOS and CaM, decreasing affinity by ∼3-fold. The rate of electron transfer from FMN is greatly attenuated; however, little effect on electron transfer from FAD is observed. The mutated proteins showed reduced FMN binding, from 20% to 60%, suggesting an influence of this residue on FMN incorporation. The weakened FMN binding may be due to conformational changes caused by the arginine mutation. Our data show that this Arg residue plays an important role in CaM binding and influences FMN binding.

AB - Nitric oxide synthases (NOSs) synthesize nitric oxide (NO), a signaling molecule, from l-arginine, utilizing electrons from NADPH. NOSs are flavo-hemo proteins, with two flavin molecules (FAD and FMN) and one heme per monomer, which require the binding of calcium/calmodulin (Ca2+/CaM) to produce NO. It is therefore important to understand the molecular factors influencing CaM binding from a structure/function perspective. A crystal structure of the CaM-bound iNOS FMN-binding domain predicted a salt bridge between R536 of human iNOS and E47 of CaM. To characterize the interaction between the homologous Arg of rat nNOS (R753) and murine iNOS (R530) with CaM, the Arg was mutated to Ala and, in iNOS, to Glu. The mutation weakens the interaction between nNOS and CaM, decreasing affinity by ∼3-fold. The rate of electron transfer from FMN is greatly attenuated; however, little effect on electron transfer from FAD is observed. The mutated proteins showed reduced FMN binding, from 20% to 60%, suggesting an influence of this residue on FMN incorporation. The weakened FMN binding may be due to conformational changes caused by the arginine mutation. Our data show that this Arg residue plays an important role in CaM binding and influences FMN binding.

KW - Calmodulin

KW - Electron transfer

KW - Flavoprotein

KW - Nitric oxide synthase

KW - Reductase

UR - https://www.scopus.com/pages/publications/84875948919

U2 - 10.1016/j.abb.2013.03.004

DO - 10.1016/j.abb.2013.03.004

M3 - Article

C2 - 23507581

AN - SCOPUS:84875948919

SN - 0003-9861

VL - 533

SP - 88

EP - 94

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

IS - 1-2

ER -

Intra- and inter-molecular effects of a conserved arginine residue of neuronal and inducible nitric oxide synthases on FMN and calmodulin binding

Abstract

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Keywords

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