Science - USA (2022-06-10)

INSIGHTS | PERSPECTIVES

ILLUSTRATION: V. ALTOUNIAN/SCIENCE; PDB DATA: ANDRÉ HOELZ AND MARTIN BECK

science.org SCIENCE

By Thomas U. Schwartz

I

n eukaryotic cells, the genome is seques-

tered in the nucleus, shielded from the

cytoplasm by the double-layered nuclear

envelope (NE). Transport of macromol-

ecules across the NE occurs through

nuclear pore complexes (NPCs), which

perforate the NE at ~200 to 2000 positions

( 1 – 3 ). Ions and molecules up to ~40 kDa dif-

fuse through NPCs, whereas larger cargo se-

lectively associate with soluble nuclear trans-

port factors to be ferried through the central

NPC channel ( 4 ). But it has been unclear how

NPCs exactly control the transport of a vast

array of different substrates, including solu-

ble proteins, embedded membrane proteins,

RNAs, and even some viral capsids. On pages

1174, 1175, 1176, 1177, and 1178 of this issue, Bl ey

et al. ( 5 ), Petrovic et al. ( 6 ), Mosalaganti et al.

( 7 ), Zhu et al. ( 8 ), and Fontana et al. ( 9 ), re-

spectively, now provide molecular structures,

in unprecedented detail, of how NPCs are

built. These findings will enable approaches

to further dissect the many NPC functions.

With an estimated size of 60 to 120 MDa,

depending on the eukaryotic species, NPCs

are elaborate protein assemblies. The 600 to

1000 individual proteins, collectively called

nucleoporins (NUPs), are organized around

a central eightfold rotational symmetry into

protomers (also sometimes referred to as

“spokes”). Protomer segments build four con-

centric rings, referencing their position rela-

tive to the pore opening in the NE: cytoplas-

mic ring (CR), inner ring (IR), nucleoplasmic

ring (NR), and luminal ring (LR). Further,

each protomer consists of subcomplexes,

which are defined as biochemically stable

entities into which NPCs disassemble dur-

ing cell division and NE breakdown. About

half of the core scaffold, which encompasses

~25 different NUPs, has previously been po-

sitioned within the NPC structure, primarily

through a combination of x-ray structural

analysis of individual subcomplexes and

cryo–electron tomography (cryo-ET) recon-

structions (or maps) of the entire NPC ( 1 – 3 ).

The papers in this issue now fill many of the

remaining gaps.

Starting from an established protocol

for the cryo-ET analysis of human NPCs,

Mosalaganti et al. improved the resolution of

the CR and IR from ~2.3 to ~1.2 nm. In addi-

tion, the authors used artificial intelligence–

based structure prediction and subcomplex

modeling to interpret the improved cryo-ET

map. The NPC scaffold also serves as the an-

chor for phenylalanine-glycine (FG) repeats

that extend their fibrillar extensions into

the central channel of the NPC to form the

principal transport barrier ( 10 ). Mosalaganti

et al. can now position, in addition to the

central NUP62-NUP58-NUP54 complex, the

second main FG-containing assembly—the

CR-attached NUP62-NUP88-NUP214 com-

plex—completing the anchor points for the

FG network.

The same cryo-ET map was also inter-

preted by Petrovic et al. and Bley et al.; these

authors used extensive experimental data

to support the fitting of experimental struc-

tures. Petrovic et al. focus on linker NUPs

that connect subcomplexes within the NPC.

They establish how the large, stacked helical

proteins NUP188 and NUP205 interact com-

petitively with NUP93. Together with a num-

ber of weaker interaction motifs, the authors

provide a linker map of the IR.

Bley et al. focus on the CR and specifically

on the attachment of so-called cytoplasmic

filaments, which are particularly important

for mRNA export. NUP358 is an extended,

multidomain protein, and five copies an-

chor to the CR through the amino-terminal

75 kDa a-solenoid element, solved by Bley et

al. by using x-ray crystallography. The flex-

ibly linked NUP93-NUP205 pair, hitherto

considered a component of the IR, is also

Massachusetts Institute of Technology, Department of

Biology, Cambridge, MA, USA. Email: [email protected]

with two chlorides. The catalyst, a urea-

sulfinamide, promoted the substitution

of only one of the chlorides by an amine

to yield a highly enriched single-handed

P-stereogenic chlorophosphonamide. This

is a highly versatile P-stereogenic build-

ing block because the chloride group and

the amino group have the orthogonal re-

activity necessary for the stereocontrolled

sequential substitution of the two groups.

Basic reagents react with inversion of con-

figuration with the chloride, whereas the

amine can be substituted under acidic

conditions for alcohols. This approach al-

lows the synthesis of a large variety of

P-stereogenic compounds, including phos-

phonates, phosphinates, phosphonami-

dates, and phosphonate thioesters. The

potential of this methodology is demon-

strated by Forbes and Jacobsen in the syn-

thesis of a few P-stereogenic drugs.

Phosphine chloride compounds are

highly reactive. To harness the reactivity

of dichlorophosphinyl derivatives in a de-

symmetrization reaction is an impressive

achievement. However, this success comes

with a caveat. The versatile chlorophosph-

inamide building block cannot be isolated

in pure form because it is prone to racemi-

zation, that is, an equilibration to a 50/50

mixture of right- and left-handed isomers.

This hampers the enrichment of the isomer

of interest and ultimately might limit the

use of this method. Future developments

in the catalytic synthesis of P-stereogenic

compounds should seek methods with im-

proved selectivity and stable P-stereogenic

intermediates that can compete with state-

of-the-art traditional chiral auxiliary ap-

proaches. The work of Forbes and Jacobsen

will encourage chemists to work on further

developments and to explore the wonders

that await us beyond the looking glass. j

R EFERENCES AND NOTES

1. K. C. Forbes, E. N. Jacobsen, Science 376 , 1230 (2022).


2. J. Meisenheimer, L. Lichtenstadt, Ber. Dtsch. Chem. Ges.
   44 , 356 (1911).


3. W. S. Knowles, Acc. Chem. Res. 16 , 106 (1983).


4. C. McGuigan et al., J. Med. Chem. 48 , 3504 (2005).


5. H. Zhang, Q.-D. You, X.-L. Xu, J. Med. Chem. 63 , 3785
   (2020).


6. K. W. Knouse et al., Science 361 , 1234 (2018).


7. S. Lemouzy, L. Giordano, D. Hérault, G. Buono, Eur. J. Org.
   Chem. 2020 , 3351 (2020).


8. D. A. DiRocco et al., Science 356 , 426 (2017).


9. A. L. Featherston et al., Science 371 , 702 (2021).

10.1126/science.abq5073

“This approach allows the

synthesis of a large variety of

P-stereogenic compounds...”

MOLECULAR BIOLOGY

Solving

the nuclear

pore puzzle

Using a battery of tools,

the architecture of

the nuclear pore complex

is revealed

1158 10 JUNE 2022 • VOL 376 ISSUE 6598