Intramembrane proteases are a family of proteases that function within cell membranes. They were discovered in 1997 and represent a new class of proteases that use different mechanisms than the traditional water-soluble protease classes. Structural studies have provided insights into their catalytic mechanisms and gating movements that allow substrate access despite operating in the low-water environment of membranes. Further research is still needed to understand aspects of intramembrane protease structure, function, and regulation.

1. Intramembrane Proteases
BSE638 Term Paper
Chintalagiri Shashank, Y5157
November 28, 2011
1 Introduction and biological significance
Proteases are of unquestioned importance to the biological machinery, be it for signalling, regulation, digestion,
etc. and have been studied for decades. The four main kinds of proteases (serine, cysteine, aspartyl and
metalloproteases) all depend on water for their protease activity and are all characteristically water-soluble.
The environment of the membrane, however, is not one where water is in abundance. It was largely expected
that proteases, atleast the kinds we know of, would not be able to function intramembrane. The discovery of
intramembrane proteases in 1997 has brought about a new phase in the study of proteases and in transmembrane
signalling mechanisms. Such proteins have been named intramembrane-cleaving proteases (I-CLiPs). The lack
of sufficent structure information from eukaryotes has lead to the study of I-CLiPs being focussed on bacterial
and archaeal analogs, and their structres are used as models. So far, I-CLiPs have been identified that belong to
the serine, aspartyl and metalloprotease classes, but the existence of cysteine I-CLiPs remains an open question.
The availability of I-CLiPs suggests a simple and irreversible strategy for signalling, which is that membrane-
tethered proteins can be cleaved to release a polypeptide in the cytosolic or extracellular domains, which can
then act as signalling molecules in their own right.
Intramembrane proteolysis was first described as an essential activity in sterol homeostasis. When cholesterol
levels in the cell drop, the cytoplasmic domain of the mammalian sterol regulatory element-binding protein
(SREBP) which is initially tethered by a transmembrane segment to the membrane of the endoplasmic reticulum
(ER) is moved to the Golgi apparatus, where it is cleaved by a transmembrane metalloprotease to release a
polypepdite domain which activates genes responsible for cholesterol and fatty acid synthesis. Since then, a
number of other I-CLiPs have been discovered. Other proteases of this family (known as S2P proteases, since
they require the substrate to be preprocessed by other enzymes) are known to be involved in responses to stress
caused by protein misfolding on the ER, or incorrect membrane assembly in the cell envelope.
Intramembrane serine proteases are generally rhomboid proteases, so names due to their structure. They are
generally involved in intercellular signalling activity rather than intracellular signalling, such as EGF related
activity in Drosophila. They are also used for a variety of other purposes in a number of organisms. In addition,
a number of similar proteins (high sequence similarity) are known to exist which do not display proteolytic
activity due to the lack of catalytic residues in the active site. Such proteins are called iRhoms and are believed
to have chaperone like activity.
A number of intramembrane aspartyl proteases are also known, but the structures of these proteins are yet
to be well established due to their being more recalcitrant. They have only been studied by electron microscopy
as of yet.
2 Structure of the Proteins
2.1 S2P (Metalloprotease)
The structure of the S2P I-CLiPs mimic their soluble metalloprotease counterparts. The specific protein whose
structure is well known is the Methanocaldococcus jannaschii mjS2P I-CLiP, which seemingly was the easiest to
obtain and crystallize. This protein consists of 6 transmembrane segments as well as one small β sheet. Of the
6 segments, 2 provide active site residues while the others support and enclose the active site region, forming a
microenvironment in which proteolysis is prossible.
Soluble metalloproteases and the matrix metalloproteases coordinate a metal ion using two histidines on
successive turns of an -helix and a third residue from a nearby helix or loop. This zinc-coordinating motif is
clearly applicable to transmembrane helices, as seen in mjS2P with His 54, His 58 and Asp 148. The glutamate
involved in water deprotonation by soluble metalloproteases also has a direct counterpart in mjS2P (Glu 55).
The mjS2P asparagine (Asn 140) that may interact with a substrate carbonyl is in a comparable position to
asparagines in the metalloproteases carboxypeptidases A and B. The proper positioning of Asn 140 in mjS2P
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2. relative to the downstream zinc-coordinating residue Asp 148 is accomplished by a break and dislocation of
the TM4 helix. Helix-breaking prolines are consistently found between the conserved asparagine and aspartate
residues in the TM4 region of S2P-family sequences.
Further, when crystallized, two conformations of the protein were trapped. From a comparision of the two
structures, it was deduced that a gating mechanism may exist in which TM1 and the TM5-TM6 pair slide apart
exposing the catalytic region to the substrate. This hypothesis is supported by the fact that there is a lack of
specific polar interactions between TM1 and TM2, allowing loose packing and hence movement, as well and
two possible salt bridge partners on TM4 corresponding to a glutamate on TM6, which may be the basis for
a comformational switch. The requirement of the catalytic water for the reaction is provided for by a narrow
channel perpendicular to the plane of the membrane. It is hypothesized that the width of this channel increases
as a side effect of the gating movement which allows the substrate into the active site.
2.2 GlpG (Serine Protease)
The example of serine I-CLiPs described is that of the bacterial I-CLiP GlpG, which consists of 5 transmembrane
segments encircling a central helix that spans only part of the membrane, creating a water-filled cavity that
is exposed to the periplasmic side of the membrane. Further, a helical hairpin between TM1 and TM2 exists
that is perpendicular to the transmembrane segments and is embedded in the outer leaflet of the membrane.
This hairpin supports the back wall of the active site and is believed to stabilize the structure. It may also be
involved in sensing the surrounding membrane environment.
The active site itself is quite different from that of soluble serine proteases like trypsin. It is formed by a serine
at the N-termius of the central α helix and a histidine is located on a parallel TM segment. While this bears
some resemblance to the active site of subtilisin, the topology of the active site is inverted when compared with
the soluble protein’s active sites, assuming the substrate enters between TM2 and TM5. The gating mechanism
for this protein is deduced similarly as above, where it is found that TM5 has the greatest variability in structure
and position. It is believed that movement of TM5 allows passage of the substrate between TM2 and TM5 into
the pocket and against TM4 which contains one half of the catalytic diad.
In addition to the catalytic dyad, a second class of conserved residue in serine I-CLiPs is the HX4HX3N
sequence from TM2. Together with a backbone NH group, this motif is in the proper position to constitute a
proton-donating pocket, the oxyanion hole, which stabilizes an intermediate formed during substrate cleavage.
One of the GlpG structures has the phosphate group of a phospholipid in this site, indicating that this pocket
may accomodate negatively charged groups.
A third class of important conserved residue is a set of glycines that allow the close approach of the three
catalytically important helices. In particular, glycines on the TM4-interacting face of two successive turns of
TM6 bring the serine and histidine of the active-site dyad into proximity, as well as being a common stabilizing
motif in membrane proteins. A glycine two residues before the catalytic serine may leave room for the oxyanion
hole. Finally, the glycine-rich segment upstream of the active-site serine in GlpG positions hydrogen-bond donor
and acceptor groups towards the cavity above the active-site serine, perhaps to bind the polypeptide backbone
N-terminal to the scissile bond.
3 Structure based functional understanding
These proteins exemplify a number of ways in which the structure has helped understand how the proteins
themselves function. To begin with, the comparision of the structures to their soluble analogues has allowed
inferences to be drawn about these relatively unknown proteins based on our understanding of typical proteases
which have been studied for decades. This is a valuable advantage we have, where we know what to look for
in the active site. Thanks to this, and looking at the other residues in the active sites, it has been possible to
make guesses as to the specificity of the proteases, such as the hypothesis that GlpG is capable of accomodating
negatively charged groups. Additionally, the analogy with soluble proteases has allowed the investigation into
synthetic substrates which will bind to the proteases to be directed. This furthers our understanding of the
specificity of the proteases in question.
Secondly, the study of the flexible regions in the structures has allowed us to make guesses about the gating
mechanism that allows a water based reaction to take place in the membrane environment, and gives us valuable
information about how the substrates enter the otherwise sealed active sites. This information would have been
otherwise very hard to obtain when taking into the fact that the structres of membrane proteins are hard to
determine to begin with.
Thirdly, structural cues have allowed the proposal of hypothesis to answer the questions of protein-lipid
interactions and the involvement of the same in substrate recognition in cleavage. Both GlpG and mjS2P have
structural protusions which are small enough to fit into a single leaflet of the membrane, which suggests that
the protein may be able to influence local lipid structure. These are a helical hairpin in GlpG and a small 4
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3. stranded β sheet in mjS2P. Study of these interactions may also lead to new ideas related to translocation of
the proteins in the membrane.
4 Unanswered questions
There are a lot of unanswered questions in the structure and function of Intramembrane Proteases, such as:
• Where are the cysteine proteases? Is there a reason for them to not work?
• How are these proteases specific? For instance, in the case of Rhomboid-I of Drosophila, the presesnce of a
positive heliz destabilizing GA motif is sufficient to make a polypeptide an efficient Rhomboid-I substrate.
• In fact, all I-CLiP seem to be particularly susceptible to unwinding of transmembrane helices. However,
none of them have ever been crystallized, so the question of why exactly this is necessary seems to be an
open one. It may have something to with limited mobility in the membrane coupled with the need for
intimate interaction with the protease at the active site.
• A number of I-CLiPs seem to select substrates only after they have been precleaved by other proteases.
The need for this is not fully understood, but this information is required to fully understand the protease
- substrate interaction. It may be that the precleavage removes water soluble parts of the substrate,
allowing the rest to be cleaved in membrane. It is also possible that a suppressor domain is removed from
the substrate aloowing it to bind and be cleaved.
• The precise role of many I-CliPs in Eukaryotes is not fully known. The full scope of signalling activity
that is possible has not been realized or characterized.
• Structures of Eukaryotic I-CLiPs have not yet been determined, and we only guess based on prokaryotic
analogues.
5 Conclusions
Intramembrane Proteases form a very interesting family of proteins. While the basic catalytic chemistry is
strikingly similar to that of their soluble counterparts, everything else about these proteins are different. They
are excellent examples of what seems to be convergent evolution, given that a number of I-CLiPs show very little
sequence similarity even within the family. As elements in intracellular and intercelular signalling, members of
this family are involved in tissue growth, necrosis, cellular and tissue environmental response, hormone response,
etc. As such, they and any specific inhibitors we may be able to synthesize are potentially interesting targets for
disease control, perhaps even in certain tumours. Of course, our understanding of these proteins as of now is far
from the stage when useful drugs may be developed. It seems that Aspartyl I-CLiPs may be crystallized in the
near future, allowing us a closer view into yet another sub-family, possibly confirming the current hypothesis
that their structure will be similar to the soluble analogues, in much the same way that the metalloprotease
ones are.
References
• How intramembrane proteases bury hydrolytic reactions in the membrane; Elinor Erez, Deborah Fass & Eitan Bibi; Nature
Vol 459, 21 May 2009;doi:10.1038/nature08146
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