Structural basis for allosteric regulation of
human phosphofructokinase-1

Phosphofructokinase-1 (PFK1) catalyzes the rate-limiting step of glycolysis,
committing glucose to conversion into cellular energy. PFK1 is highly regu
lated to respond to the changing energy needs of the cell. In bacteria, the
structural basis of PFK1 regulation is a textbook example of allostery; mole
cular signals of low and high cellular energy promote transition between an
active R-state and inactive T-state conformation, respectively. Little is known,
however,aboutthestructural basis for regulation of eukaryotic PFK1. Here, we
determine structures of the human liver isoform of PFK1 (PFKL) in the R- and
T-state by cryoEM, providing insight into eukaryotic PFK1 allosteric regulatory
mechanisms. The T-state structure reveals conformational differences
between the bacterial and eukaryotic enzyme, the mechanisms of allosteric
inhibition by ATP binding at multiple sites, and an autoinhibitory role of the
C-terminus in stabilizing the T-state. We also determine structures of PFKL
f
ilaments that define the mechanism of higher-order assembly and demon
strate that these structures are necessary for higher-order assembly of PFKL
in cells.

Glycolysis is an ancient, highly-conserved metabolic pathway for the
extraction of energy from sugars. During glycolysis, glucose is meta
bolized to produce energy in the form of ATP, the essential cofactor
NADH, as well as other biosynthetic precursors to support cellular
functions. The first committed step of glycolysis is catalyzed by
phosphofructokinase-1 (PFK1), which converts fructose 6-phosphate
(F6P) to fructose 1,6-bisphosphate (F1,6BP), consuming one molecule
of ATP in the process. Given this central role as the gatekeeper of
glycolysis, PFK1isheavilyregulatedbytheenergystateofthecell;PFK1
is activated by signalsof lowcellularenergy, such as AMPandADP,and
inhibited by signals of high cellular energy, such as ATP and citrate.

cterial enzyme1–3. Bacterial PFK1 is a D2-symmetric homotetramer
with four active sites, each formed at an interface between two
monomers. The enzyme transitions between an active R-state con
formation, promoted by binding to F6P and allosteric activators, and
an inactive T-state conformation, observed in the absence of F6P and
upon binding to allosteric inhibitors. The R-state to T-state transition
involves a rotation between essentially rigid dimers and rearrange
mentofactivesiteresidues,whichtogetherfunctiontodisrupttheF6P
binding pocket2.

The PFK1 catalytic domain architecture is conserved in eukar
yotes. However, eukaryotic PFK1 has an additional regulatory domain,
which arose from gene duplication, tandem fusion, and evolution of
the ancestral prokaryotic catalytic domain4–6. The resulting eukaryotic
PFK1 monomer corresponds to the bacterial dimer that rotates as an
essentially rigid body during the R- to T-state transition. Generally,
eukaryotic PFK1 tetramerizes via its regulatory domains, producing a
quaternary structure distinct from that observed for the prokaryotic
enzyme7,8. Oligomerization regulates PFKL activity; tetramers with
D2 symmetry represent the active form of the enzyme, and allosteric
activators promote tetramerization, while allosteric inhibitors pro
mote tetramer disassembly7,9–11. Eukaryotic PFK1 is subject to addi
tional layers of regulation, being regulated by over 20 allosteric　 ligands12, in addition to post-translational modification by phosphor
ylation, glycosylation, and acetylation13–16. Vertebrates possess three
PFK1 isoforms: platelet (PFKP), muscle (PFKM), and liver (PFKL), each
with particular catalytic and regulatory properties, as well as tissue
specificexpressionprofiles17.PFKLformsfilamentouspolymersinvitro
and micron-scale puncta in cells18. However, whether higher-order
assemblyofPFKLincellsreflects the filamentformationobservedwith
purifiedproteinisunclear. Further, the functionalrole ofthesehigher
order assemblies remains an open question.
Bacterial PFK1providesacanonicalexampleofthestructuralbasis
for allosteric regulation, though little is known about the structural
basis for the regulation of the vertebrate enzyme. Existing crystal
structures of vertebrate PFK1 in various ligand states all resemble the
R-state, suggesting that crystal packing may preferentially select for
the R-state conformation6–8.
Here, we determine cryoEM structures of PFKL in the R- and
T-state conformations. The conformation of T-state PFKL differs from
its bacterial counterpart and other vertebrate PFK1 structures. The
T-stateconformationofPFKLisstabilizedbybindingoftheC-terminus
across the catalytic and regulatory domains, and truncation of the
C-terminusdisruptsPFKLregulation.WefurthershowthatPFKLforms
f
ilaments in both the R- and T-state conformations, present cryoEM
structures of filaments in both states, and demonstrate that micron
scale, punctate assemblies of PFKL observed in cells are composed of
the same filament structures observed in vitro.

PFK‑1 における ATP の結合部位は、各サブユニットの“中央の深い溝（cleft）”にある ATP‑binding pocket で、F6P 結合部位に隣接した 二重基質ポケット に位置します。
この位置は構造解析でも明確に示されており、ATP は Mg²⁺ と複数の正電荷残基によって固定されます Nature EMBL-EBI。

PFK‑1 の ATP 結合部位（Catalytic ATP-binding site）

◆ ATP が結合する場所（構造的特徴）

• サブユニット内部の中央溝（deep cleft）
• F6P 結合部位のすぐ隣
• 二重基質ポケット（dual-substrate pocket）の一角
• R-state（活性型）で開き、T-state（不活性型）で閉じる領域

Cryo-EM と X線構造解析で、この ATP-binding pocket が 活性中心の一部として固定されていることが示されています Nature。

ATP 結合に関わる主要残基（E. coli PFK‑1, PDB: 1PFK）

ATP が“どこに”結合するかを一言で言うと

PFK‑1 の ATP は、各サブユニットの中央溝にある二重基質ポケットに結合し、Mg²⁺と正電荷残基（Arg72/Arg172）によって固定される。

これは F6P と隣り合う位置で、
Asp128 が F6P を脱プロトン化 → F6P が ATP の γリン酸を攻撃
という反応が起こるため、ATP は必ずこの位置に来ます。

必要なら、
ATP-binding pocket と allosteric ATP-binding site（阻害部位）の違い

も整理できます。