B.S. College of Mount St. Vincent, 1996
Ph.D. Biochemistry & Structurial Biology, 2012
Year Medical Student
Steven O. Smith, Ph.D.
Department: Biochemistry & Cell Biology
Program: Biochemistry & Structural Biology
Title: The role of the transmembrane domain in the
structure and function of the cytokine receptor family
Activation of the erythropoietin receptor (EpoR) by the soluble cytokine
erythropoietin (Epo) is essential for the differentiation of erythrocyte
progenitors and their development into red blood cells. The single transmembrane
(TM) helix of the EpoR mediates dimerization of the receptor in the
inactive state and is responsible for coupling ligand binding to activation
of an intracellular Janus kinase. Neither the structure of the inactive
dimer nor the structural changes in the TM region that occur upon ligand
binding are known. This work presents the solution NMR structures of
peptides corresponding to the TM and juxtamembrane (JM) sequences that
bridge the extracellular and intracellular domains. The N terminal end
of the TM-JM peptides (LTASDLDPLILT) contains the transition point between
the last b-strand of the extracellular D2 domain of the receptor and
the TM a-helix. NMR measurements indicate that the TM helix extends
to Leu226. The proline residue allows Asp224 to fold back and form side
chain hydrogen bonds to the backbone NH of Leu226. Structural studies
on the TM region of EpoR alone reveal intermolecular contacts between
polar residues (Ser231, Ser238 and Thr242). At the intracellular TM-JM
boundary, the defined a-helical secondary structure appears to break
at the Arg250-Arg251 sequence. However, Leu253-Lys256 exhibit downfield
carbonyl chemical shifts consistent with helical structure for the JM
switch region. To stabilize the TM-JM peptides in an active conformation,
two approaches were undertaken. The first approach was to substitute
Leu223 with cysteine. The full length L223C mutant of EpoR was previously
shown to have constitutive activity. The second approach was to characterize
the complex between the TM-JM dimer and the TM domain of an EpoR-activating
viral membrane protein, Gp55-P. In both cases, the largest chemical
shift changes were at the intracellular TM-JM boundary, particularly
His249. A mechanism of receptor activation that unites biophysical and
biochemical data is presented.
(pre-MSTP publications indicated with an *)
Itaya M, Brett IC, Smith SO. (2012). Synthesis,
purification, and characterization of single helix membrane peptides
and proteins for NMR spectroscopy. Methods Mol Biol.
J, Defour JP, Pecquet C, Leroy E, Antoine-Poirel H, Brett I,
Itaya M, Smith SO, Vainchenker W, Constantinescu SN. (2011). Orientation-specific
signalling by thrombopoietin receptor dimers. EMBO J.
30(21):4398-413. doi: 10.1038/emboj.2011.315. PMID:21892137.
BE, Brett IC. (2009). Mouse models of influenza. Handbook
on Immunosenescence: basic understanding and clinical applications.
Fulop, T.; Franceschi, C.; Hirokawa, K.; Pawelec, G. (Eds.) Springer
Science+Business Media BV.
Johansson BE, Brett
IC. (2008). Recombinant influenza B virus HA and NA antigens
administered in equivalent amounts are immunogenically equivalent and
induce equivalent homotypic and broader heterovariant protection in
mice than conventional and live influenza vaccines. Human
Mohiyiddeen G, Brett
I, Jude E. (2008). Infective endocarditis caused by taphylococcus
aureus in a patient with atopic dermatitis: a case report. J
Med Case Rep. 2:143. PMID:18454875.
Johansson BE, Brett IC. (2007). Changing perspective
on immunization against influenza. Vaccine.
*Brett IC, Johansson BE. (2006). Variation of divalent
cation requirements of influenza A virus N1 neuraminidases. J
BE, Brett IC. (2006). An inactivated subvirion influenza
A (H5N1) vaccine. NEJM. 354(25):2724-5. (Letter
to the editor)
*Muddu BN, Umaar R, Kim WY, Zenios M, Brett I, Sharma Y. (2005). Whiplash
injury of the shoulder: is it a distinct clinical entity? Acta
Orthop Belg. 71(4):385-7. PMID:16184990.
*Brett IC, Johansson BE. (2005). Immunization against
Influenza A virus: Comparison of conventional inactivated, live-attenuated,
and recombinant baculovirus produced purified hemagglutinin and neuraminidase
vaccines in a murine model system. Virology.
A, Yahalom J, Zaretsky L, Brett I, Zelenetz AD. (2005).
Gastric mucosa-associated lymphoid tissue detected by clonotypic polymerase
chain reaction despite continuous pathologic remission induced by involved-field
radiotherapy. J Clin Oncol. 23(16):3768-72.
ED, Pokorny BA, Johansson B, Brett I, Milev Y, Matthews
JT. (2004). Protection of mice with recombinant influenza virus neuraminidase.
J Infect Dis. 189(3):459-61.
*Johansson, BE and
Brett, IC. (2003). Variation in divalent cation requirements
of influenza A virus N2 neuraminidases. J Biochem.
ST, Brett I, Almenoff PL, Lesser M, Terrin M, Teirstein
AS. (2003). Recovery of cell wall deficient organisms from blood does
not distinguish between subjects with sarcoidosis and controls. Chest.
ED, Smith C, Brett I, Pokorny BA, Johansson B, Cox
N. The total influenza vaccine failure of 1947 revisited: major intrasubtypic
antigenic change can explain failure of vaccine in a post-World War
II epidemic. Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10748-52. Epub
2002 Jul 22. Erratum in: Proc Natl Acad Sci U S A. 2003 Jan 21;100(2):764.
I, Werber J, Kilbourne ED. (2002). Rapid confirmation by RFLP
of transfer to vaccine candidate reassortment viruses of the principal
‘high yield’ gene of influenza A viruses. J
Virol Meth. 100:133-140.
ED, Smith C, Brett I, Pokorny BA, Johansson B, Cox
N. (2002). The total influenza vaccine failure of 1947 revisited: Major
intrasubtypic antigenic change can explain failure of vaccine in a post-World
War II epidemic. Proc Nat Acad Sci. 99(16):10748-10752.