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We thank our patients for their courage and generosity. We
are grateful to C. Blair and K. Judge for expert technical and
administrative assistance. We thank H. Ren, J. Olson, M. Hathcok,
H. Zeh, A. Singhi, S. Crippa, M. Ryan, and L. Ryan for their assistance
with this study. This work was supported by the Lustgarten
Foundation for Pancreatic Cancer Research; The Virginia and
D. K. Ludwig Fund for Cancer Research; The Conrad N. Hilton
Foundation; The Commonwealth Fund; The John Templeton
Foundation; the Clinomics Program; Mayo Clinic Center for
Individualized Medicine; the Mayo Clinic Biobank; The Sol Goldman
Center for Pancreatic Cancer Research; The Michael Rolfe Pancreatic
Cancer Research Foundation; The Benjamin Baker Scholarship; The
Gray Foundation; S. Wojcicki and D. Troper; The Marcus Foundation;
The Honorable Tina Brozman Foundation; The Burroughs Wellcome
Career Award for Medical Scientists; The Doris Duke Charitable
Foundation (2014107); and National Institutes of Health grants P50-
CA62924, P50-CA102701, CA06973, CA152753, GM-07309,
U01CA200469, and U01CA152753. N.P., S.Z., K. W.K., L.D., and B.V.
are founders of Personal Genome Diagnostics and PapGene.
B.V. and K. W.K are on the Scientific Advisory Board of Sysmex-Inostics. B.V. is also on the Scientific Advisory Boards of Exelixis
GP. R.H.H. is on the Board of Directors of MiDiagnostics. These
companies and others have licensed technologies from Johns Hopkins,
and N.P., K. W.K., L.D., B.V, and R.H.H. receive equity or royalties
from these licenses. The terms of these arrangements are being
managed by Johns Hopkins University in accordance with its
conflict of interest policies. L.D. is on the Board of Directors of
Jounce Therapeutics and is a Scientific Advisor for Genocea, Cell
Design Labs, and Merck. A.S.M. is a consultant for Abbvie,
Genentech, Bristol-Myers Squibb, and Trovagene. B.V., N.P., and
K. W.K. are inventors on a patent (U.S. 20140227705 A1) held by
Johns Hopkins University that covers basic aspects of the
SafeSeqS technology used in this paper. B.V., K. W.K., N.P., J.D.C.,
C. T., and A.M.L. are inventors on a patent application to be
submitted by Johns Hopkins University that covers other aspects
of SafeSeqS as well as the multi-analyte approach described in this
paper. All data needed to evaluate the conclusions in the paper
are present in the paper and/or the supplementary materials.
Contact C. T. for questions about the algorithms; A.M.L. for
questions about clinically related issues; K. W.K about the
sequencing analyses; B.V. about experimental procedures; and
N.P. about the overall design of the study.
Material and Methods
Figs. S1 to S4
Tables S1 to S11
31 October 2017; accepted 8 January 2018
Published online 18 January 2018
Structural principles that enable
oligomeric small heat-shock protein
paralogs to evolve distinct functions
Georg K. A. Hochberg,1*† Dale A. Shepherd,1*‡ Erik G. Marklund,1*§
Indu Santhanagoplan,2|| Matteo T. Degiacomi,1¶ Arthur Laganowsky,1#**††
Timothy M. Allison,1 Eman Basha,2‡‡ Michael T. Marty,1‡‡ Martin R. Galpin,1
Weston B. Struwe,1 Andrew J. Baldwin,1 Elizabeth Vierling,2 Justin L. P. Benesch1§§
Oligomeric proteins assemble with exceptional selectivity, even in the presence
of closely related proteins, to perform their cellular roles. We show that most proteins
related by gene duplication of an oligomeric ancestor have evolved to avoid
hetero-oligomerization and that this correlates with their acquisition of distinct
functions. We report how coassembly is avoided by two oligomeric small heat-shock
protein paralogs. A hierarchy of assembly, involving intermediates that are populated
only fleetingly at equilibrium, ensures selective oligomerization. Conformational
flexibility at noninterfacial regions in the monomers prevents coassembly, allowing
interfaces to remain largely conserved. Homomeric oligomers must overcome the
entropic benefit of coassembly and, accordingly, homomeric paralogs comprise fewer
subunits than homomers that have no paralogs.
Many proteins associate into selective homo- or heteromers in order to function (1). New assemblies are most often created by gene duplication of a preexisting ho- momer (2). The resulting oligomeric para-
logs initially coassemble because both have the
same sequence (and hence structure and interfaces)
as their ancestor (Fig. 1A) (3). This coassembly
can easily become entrenched if evolution of the
two resulting duplicates is functionally constrained
to maintain the interaction (4, 5), implying that
heteromerization should be the most likely fate of
oligomeric paralogs. However, when we analyzed
the human, Arabidopsis, yeast, and Escherichia coli
interactomes (supplementary materials and data
file S1), we found that most oligomeric paralogs
do not form heteromers (i.e., do not coassemble)
(Fig. 1B), despite overlapping localization and
expression profiles (fig. S1, A and B). Moreover,
we found that those paralogs that cannot co-
assemble share lower sequence identity and fewer
common functions than paralogs that can (Fig. 1,
C and D). This suggests that heteromerization
acts as a constraint on the functional divergence
of oligomeric paralogs (6). Relieving this con-
straint is therefore a key step in the evolutionary
trajectories of oligomeric proteins toward evolv-
ing new functions.
To investigate how this occurs, we examined
the selective assembly of two paralogous small
heat-shock proteins (sHSPs), molecular chaperones found across the tree of life that are key to
the cell’s ability to respond to stress (7, 8). A
duplication event led to land plants having two
classes of cytosolic sHSPs (class 1 and 2; Fig. 1E
and fig. S2) that both assemble as dodecamers
but cannot form heteromers between classes (9).
Both are required for thermotolerance in vivo
(10) and have different mechanisms of action
(11, 12). We chose one paralog of each class from
Pisum sativum: HSP18.1 and HSP17.7 (hereafter
WT-1 and WT-2, respectively). Both proteins comprise an N-terminal region, an a-crystallin domain,
and a C-terminal tail, and both form homo-12-
mers (12) using three independent interfaces:
The a-crystallin domain mediates the formation
of an isologous a·a dimer; these dimers assemble
into oligomers through heterologous contacts between the a-crystallin domain and the C-terminal
tails from neighboring dimers (a·C), and interactions between the N-terminal regions (N·N) (Fig.
1F) (13). Their complex, multi-interface architecture
makes these proteins an ideal system to investigate how evolution acts to regulate the biophysical
properties of oligomers to develop a set of selective
interfaces that allows them to diverge functionally.
Small-angle x-ray scattering experiments indicated that both proteins form tetrahedral oligomers (fig. S3), implying that there are no major
1Physical and Theoretical Chemistry Laboratory, Department
of Chemistry, University of Oxford, Oxford OX1 3QZ, UK.
2Department of Biochemistry and Molecular Biology,
University of Massachusetts, Amherst, MA 01003, USA.
*These authors contributed equally to this work. †Present address:
Department of Ecology and Evolution, University of Chicago,
Chicago, IL 60637, USA. ‡Present address: Waters Corporation,
Stamford Avenue, Wilmslow SK9 4AX, UK. §Present address:
Department of Chemistry–BMC, Uppsala University, Box 576, 75123,
Uppsala, Sweden. ||Present address: Department of Plant Sciences,
University of Cambridge, Cambridge CB2 3EA, UK. ¶Present address:
Department of Chemistry, Durham University, South Road, Durham
DH1 3LE, UK. #Present address: Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M
Health Science Center, Houston, TX 77030, USA. **Present address:
Department of Chemistry, Texas A&M University, College Station,
TX 77842, USA. ††Present address: Department of Microbial
Pathogenesis and Immunology, College of Medicine, Texas A&M Health
Science Center, Bryan, TX 77807, USA. ‡‡Present address:
Department of Chemistry and Biochemistry, University of Arizona,
1306 East University Boulevard, Tucson, AZ 85721, USA.
§§Corresponding author. Email: firstname.lastname@example.org