All-Atom Molecular Dynamics Simulations of Polyethylene Glycol (PEG) and LIMP‑2 Reveal That PEG Penetrates Deep into the Proposed CD36 Cholesterol-Transport Tunnel

Polyethylene glycol
(PEG) is the most prominent clinically administered
synthetic polymer. For example, over 300 million people have been
administered PEGylated liposome vaccines for SARS-CoV-2. PEG is used
in mammals because it has low affinity for most proteins and vice
versa. However, this makes it difficult to study the few interactions
with proteins that PEG has. On the atomistic level, there are two
PEG-protein structures: (1) PEG-LIMP-2 and (2) PEG-αPEG. In
the first structure, two monomers of a 1.5 kDa PEG polymer (PEG2)
had electron density deep in the postulated cholesterol transport
tunnel of LIMP-2, a lysosomal cholesterol transport protein and member
of the CD36 super family of proteins. It is unclear how PEG entered
this tunnel. In the second structure, PEG wrapped around a surface-exposed
tryptophan on its antibody. Since tryptophan is a rare residue, it
is unclear if this PEG-Trp interaction is ubiquitous. To gain deeper
mechanistic insight into PEG–protein interactions, we surrounded
the LIMP-2 apo structure with 13 PEG chains of 10 monomers each (PEG10),
water, and KCl and simulated the system using NAMD. One of the 13
chains penetrated LIMP-2 and came within 3 Å of PEG2. This was
possible because of the strong hydrogen bonding between multiple oxygens
along PEG10 and Arg192 but, most importantly, the clamping of the
tertiary structure on PEG10. Clamping stabilized the movements of
PEG10, and the leading oxygen of PEG10 was able to penetrate LIMP-2
and head toward to the position occupied by PEG2. Phe383 appears to
act as a gate for objects to move through this cavity, which continues
to the basal/membrane side of LIMP-2. Of all residues, PEG10 molecules
had the most sustained interactions with lysine and arginine because
of their strong hydrogen-bonding capabilities. These results show
that the oxygens of PEG bind residues with high hydrogen bonding capabilities.
However, the PEG–protein interaction is likely to be transient
unless groups of resides can clamp down on PEG or a cavity that at
least part of the PEG chain can enter is in close proximity to lower
PEG’s entropy.

聚乙二醇（Polyethylene glycol, PEG）是目前临床应用最广泛的合成聚合物。例如，已有超过3亿人次接种了针对新型冠状病毒（SARS-CoV-2）的PEG化脂质体疫苗。PEG之所以可应用于哺乳动物体内，是因为其与绝大多数蛋白质的亲和力极低，反之蛋白质对PEG同样亲和力微弱；但这一特性也使得研究PEG与蛋白质间为数不多的相互作用变得困难。
在原子尺度层面，目前已报道两种PEG-蛋白质复合物结构：(1) PEG-LIMP-2复合物；(2) PEG-αPEG复合物。在第一种结构中，分子量1.5 kDa的PEG聚合物（PEG2）的两条单体，在溶酶体胆固醇转运蛋白LIMP-2（CD36蛋白超家族成员）的假定胆固醇转运隧道深处观测到电子密度。目前尚不清楚PEG是如何进入该隧道的。在第二种结构中，PEG包裹了抗体表面暴露的色氨酸残基。由于色氨酸属于稀有氨基酸残基，因此尚不清楚这种PEG-色氨酸相互作用是否具有普遍性。
为深入解析PEG与蛋白质相互作用的机制，我们将LIMP-2的空载蛋白结构（apo结构）周围加入13条各含10个单体的PEG链（PEG10）、水及氯化钾（KCl），并使用纳米分子动力学软件（NAMD）对该体系进行模拟。13条PEG链中有1条穿透了LIMP-2蛋白，并距离PEG2的结合位置仅3埃以内。这一过程得以实现，主要依赖于PEG10链上多个氧原子与精氨酸192（Arg192）之间形成的强氢键作用，更关键的是蛋白质的三级结构对PEG10链形成了夹持锁定。这种夹持稳定了PEG10的运动，使得PEG10的前端氧原子得以穿透LIMP-2并朝向PEG2原本占据的位置移动。苯丙氨酸383（Phe383）似乎充当了该腔体的门户，该腔体延伸至LIMP-2的基底/膜侧区域。在所有氨基酸残基中，PEG10分子与赖氨酸和精氨酸的相互作用最为持久，这得益于这两种残基较强的氢键形成能力。
上述结果表明，PEG的氧原子可与具有强氢键结合能力的残基相结合。然而，PEG与蛋白质的相互作用通常是瞬时的，除非有一组残基能够夹持住PEG链，或是存在一个可供PEG部分链段进入的空腔紧邻该结合位点，以降低PEG的熵。