Macromolecular assemblies, such as a microtubule shown to the right
(Structure 2002), perform many critical functions in a cell.
Structural information is required for understanding at the
molecular and chemical levels how these machines work.
However, their large sizes make structure determination by
NMR and X-ray crystallography difficult. Thanks to the
advent of direct electron detector and novel computational
image reconstruction algorithm, single particle cryo-electron
microscopy (cryo-EM) has become a high-resolution tool for
studying structure and conformational dynamics of protein
complexes. Our lab has been using cryo-EM combined with
other biophysical and biochemical methods to study the
eukaryotic DNA replication system and the Mycobacterium
tuberculosis Pup-proteasome system over the past decade.
Eukaryotic DNA replication:
The origin recognition complex (ORC) is a six-protein
ATPase machine conserved in all eukaryotes. The yeast ORC
constitutively binds to and marks the replication origin
throughout the cell cycle. Licensing of the DNA replication
origin starts in G1 phase when the cell division cycle
protein Cdc6 binds to ORC. In collaboration with Dr. Bruce
Stillman and Dr. Christian Speck, we have revealed by cryo-EM
that ORC has a bi-lobed half-ring architecture, and that
Cdc6 completes the ring for subsequently loading of the
replicative helicase (NSMB 2005; PNAS 2008). ORC-Cdc6 is the
active loading platform that recruits the cdt1-bound Mcm2-7
hexamer one at a time (NSMB 2013), and that one ORC-Cdc6,
not two, recruits two Mcm2-7 hexamers to form the inactive
double-hexamer encircling dsDNA (G&D 2014). At G1/S
transition, the double-hexamer is converted to two active
helicases the Cdc45-Mcm2-7-GINS (CMG) complexes.
In S phase, the active CMG helicase (CMG) works with the
leading strand polymerase epsilon, the lagging strand
polymerase delta, and the primase-polymerase alpha to
synthesize new DNA. In collaboration with Dr. Michaeal
O’Donnell, we have found the leading strand polymerase
epsilon rides ahead of the helicase, rather than trailing
behind the helicase as widely believed (NSMB 2015).
The architecture of the DNA replication
origin recognition complex in Saccharomyces
cerevisiae. Chen Z, Speck C, Wendel P, Tang
C, Stillman B, Li H. Proc Natl Acad Sci U S
A. 2008, 105, 10326-31.
The origin recognition complex: a
biochemical and structural view. Li H,
Stillman B. Subcell Biochem. 2012, 62,
Cryo-EM structure of a helicase loading
intermediate containing ORC-Cdc6-Cdt1-MCM2-7
bound to DNA. Sun J, Evrin C, Samel SA,
Fernández-Cid A, Riera A, Kawakami H,
Stillman B, Speck C, Li H. Nat Struct Mol
Biol. 2013, 20, 944-51.
Structural and mechanistic insights into
Mcm2-7 double-hexamer assembly and function.
Sun J, Fernandez-Cid A, Riera A, Tognetti S,
Yuan Z, Stillman B, Speck C, Li H. Genes
Dev. 2014, 28, 2291-303.
The architecture of a eukaryotic
replisome. Sun J, Shi Y, Georgescu RE, Yuan
Z, Chait BT, Li H, O’Donnell ME. Nature
structural & molecular biology. 2015; 22,
Mycobacterium tuberculosis (Mtb) Pup-proteasome
Tuberculosis kills 1.5-2 million people globally every year. An effective
vaccine or chemotherapy has yet to be developed. Recently,
through a large-scale transposon mutagenesis screening, the
Mycobacterium tuberculosis (Mtb) proteasome and Mtb
proteasomal ATPase (Mpa) were found to be required for Mtb
resistance to killing by a source of nitric oxide (NO). NO
is required by the host immune system to control Mtb
infections. Proteasome and Mpa appear to protect Mtb against
NO by degrading proteins after exposure to NO. Thus, Mpa and
the Mtb proteasome may be promising targets for the
development of anti-Tb chemotherapeutics. We have combined
cryo-EM, X-ray crystallography, and protein biochemistry to
elucidate the structure and function of the Mtb proteasome,
Mpa ATPase, respectively. We found that the Mtb proteasome
and the associated ATPase are structurally similar to their
eukaryotic counterparts yet possess unique assembly and
gating mechanism (Structure 2009, EMBO J 2010). We
elucidated the structure basis for species-specific
inhibition of the Mtb proteasome inhibitor Oxathiazol-2-ones
(Nature 2009). We further revealed that the protein
degradation tag Pup, a prokaryotic ubiquitin-like protein,
is intrinsically disordered, but folds into an α-helix upon
binding to and recognized by the proteasomal ATPase (NSMB
2010). Recently, Heran Darwin lab discovered an
ATP-independent Mtb proteasomal activator PafE, which
surprisingly formed a dodecameric ring, as demonstrated by
EM (PNAS 2015). Our work may help structure-based anti-TB
chemotherapeutic development targeting the Pup-proteasome
Inhibitors selective for mycobacterial
versus human proteasomes. Lin G, Li D, de
Carvalho LP, Deng H, Tao H, Vogt G, Wu K,
Schneider J, Chidawanyika T, Warren JD, Li
H, Nathan C. Nature. 2009, 461, 621-6.
Structural insights on the Mycobacterium
tuberculosis proteasomal ATPase Mpa. Wang T,
Li H, Lin G, Tang C, Li D, Nathan C, Darwin
KH, Li H. Structure. 2009, 17, 1377-85.
Structural basis for the assembly and
gate closure mechanisms of the Mycobacterium
tuberculosis 20S proteasome. Li D, Li H,
Wang T, Pan H, Lin G, Li H. EMBO J. 2010,
Binding-induced folding of prokaryotic
ubiquitin-like protein on the Mycobacterium
proteasomal ATPase targets substrates for
degradation. Wang T, Darwin KH, Li H. Nat
Struct Mol Biol. 2010, 17, 1352-7.
An adenosine triphosphate-independent
proteasome activator contributes to the
virulence of Mycobacterium tuberculosis.
Jastrab JB, Wang T, Murphy JP, Bai L, Hu K,
Merkx R, Huang J, Ovaa H, Gygi SP, Li H,
Darwin KH. Proc Natl Acad Sci U S A. 2015;
Membrane-embedded enzyme complexes
Membrane proteins, in particular eukaryotic membrane
proteins, are underrepresented in the protein structural
database. This is so because it is often difficult to
produce sufficient material for traditional protein
crystallography, and membrane complex is generally sensitive
to the detergents used for solubilization and purification.
Cryo-EM is uniquely suited for structural analysis of
membrane complexes, as minimum amount of material is
required and the method is compatible with many mild
detergents. We have analyzed the structures of the bacterial
pilus assembly ushers (Cell 2008, Nature 2011, NSMB 2015),
the yeast oligosaccharyl transferase complex that N-glycosylates
the nascent polypeptide chains (Structure 2008), and the
ER-anchored Xxylt1 that O-glycosylates the Notch receptor
(Nat Chem Bio 2015).
Fiber formation across the bacterial
outer membrane by the chaperone/usher
pathway. Remaut H, Tang C, Henderson NS,
Pinkner JS, Wang T, Hultgren SJ, Thanassi
DG, Waksman G, Li H. Cell. 2008, 133,
Structure of the oligosaccharyl
transferase complex at 12 A resolution. Li
H, Chavan M, Schindelin H, Lennarz WJ, Li H.
Structure. 2008, 16, 432-40.
Crystal structure of the FimD
usher bound to its cognate FimC-FimH
substrate. Phan G, Remaut H, Wang T, Allen
WJ, Pirker KF, Lebedev A, Henderson NS,
Geibel S, Volkan E, Yan J, Kunze MB, Pinkner
JS, Ford B, Kay CW, Li H, Hultgren SJ,
Thanassi DG, Waksman G. Nature. 2011, 474,
The pilus usher controls protein
interactions via domain masking and is
functional as an oligomer. Werneburg GT,
Henderson NS, Portnoy EB, Sarowar S,
Hultgren SJ, Li H, Thanassi DG. Nat Struct
Mol Bio. 2015, 22, 540-6.
structures support an SNi-like retaining
mechanism. Yu H, Takeuchi M, LeBarron J,
Kantharia J, London E, Bakker H, Haltiwanger
RS, Li H, Takeuchi H. Nat Chem Bio. 2015,
One of ten national laboratories overseen and primarily funded by the Office of Science of the
U.S. Department of Energy (DOE), Brookhaven National Laboratory conducts research in the physical,
biomedical, and environmental sciences, as well as in energy technologies and national security.
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and government researchers. Brookhaven is operated and managed for DOE's Office of Science by Brookhaven
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University of New York on behalf of Stony Brook University, the largest academic user of Laboratory
facilities, and Battelle, a nonprofit applied science and technology organization.