Finding function through structural genomics Lawrence Shapiro* and Tim Harris
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btb107.qxd 02/16/2000 03:20 Page 31
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Finding function through structural genomics
Lawrence Shapiro* and Tim Harris†
The recent availability of whole-genome sequences and large kinases had been well documented before the first crystal
numbers of protein-coding regions from high-throughput cDNA structure revealed the molecular details of their function
analysis has fundamentally changed experimental biology. [6]. And the functions of the molecular chaperones GroEL
These efforts have provided huge databases of protein [7] and DnaK [8] were well understood before structural
sequences, many of which are of unknown function. studies revealed the details of their function.
Deciphering the functions of these myriad proteins presents a
major intellectual challenge. Is this detail-uncovering role the limit of what structural
studies can give to biology? We think that structural stud-
Addresses ies can now be gainfully employed as part of early phase
*Structural Biology Program, Department of Physiology and biological discovery, to answer the question ‘what is the
Biophysics, Mount Sinai School of Medicine, 1425 Madison Avenue, function of this protein?’ This is the defining feature of
New York, NY 10029, USA;
e-mail: shapiro@anguilla.physbio.mssm.edu
structure-based functional genomics.
† Structural Genomix Inc., 10505 Roselle Street, San Diego, CA
92121, USA; e-mail: tim@stromix.com Almost universally, when a new cDNA sequence is deter-
mined, it is compared by basic local alignment search tool
Current Opinion in Biotechnology 2000, 11:31–35
(BLAST) searching with cDNAs corresponding to proteins
0958-1669/00/$ — see front matter © 2000 Elsevier Science Ltd. of known function [9]. A similarity ‘hit’ is often used to cat-
All rights reserved. egorize the biological function of the protein deriving from
Abbreviations that cDNA: a kinase-related sequence is usually thought of
BLAST basic local alignment search tool as a kinase, and biological studies on such a molecule
HIT histidine triad would generally begin with the overall idea to look for
PTB phosphotyrosine binding
SH2 src homology-2
kinase function. Three-dimensional (3D) structures, how-
TNF tumor necrosis factor ever, provide advantages over sequence data in two
distinct areas. Firstly, 3D structural information can pro-
vide new insights into biology by elucidating relatedness
Introduction to proteins of known biological function, which could not
While the revolution in genomics has provided compre- have been arrived at by sequence analysis alone. Secondly,
hensive lists of newly discovered proteins, a technological structural information can identify binding motifs and cat-
revolution in crystallography (particularly multiwavelength alytic centers — even for proteins without a known
anomolous diffraction [MAD] analysis [1,2] and the advent biological function. These, to our minds, are the two main
of new synchrotron sources enabling the use of undulator principles of the structure-based functional genomics
radiation [3,4•]) has enabled a quantum leap in the rapidi- approach. Here we present a brief review on the current
ty with which structures can be determined. It is now state of structure-based functional genomics, which uses
therefore possible to determine high-resolution structures protein structure as a cornerstone in a strategy for deter-
of proteins with greatly improved efficiency. The central mining protein function.
question addressed in this review is this: can structural
knowledge enable us to efficiently answer early-stage bio- Aha! Functional relationships derived through
logical questions of protein function? Structural biology structural similarity
has focused mainly on the details of protein function, ask- Sequence conservation is, in general, much weaker than
ing the question ‘how does the protein achieve its structural conservation. A good example of this can be seen
function?’ Can we now use protein structure to answer the in comparing the oxygen carriers from blood and muscle,
more basic question ‘what does the protein do?’ hemoglobin and myoglobin. Although these proteins are
not clearly related in sequence (e.g. a BLAST search with
Traditionally, structural studies have come near the ‘end’ hemoglobin will not elicit a myoglobin ‘hit’), they are
of the biological discovery process. After the function of a nonetheless closely related in 3D structure [10]. Thus, we
protein or protein family has been well characterized by can imagine the hypothetical situation of discovering myo-
biochemistry, cell biology, and molecular biology studies, globin for the first time as a muscle-specific cDNA, but not
the crystal structure has been a logical ‘ultimate’ step for knowing its function. Determining its 3D structure and
understanding the atomic bases of these molecular func- comparison to the database of known proteins might lead
tions. Examples of this approach are almost too numerous us to exclaim: ‘Aha! This must be an oxygen carrier!’
to mention, for it includes most of structural biology:
trypsin was known to be a protease, and its specificity and A real world example of this is illustrated in a study of
kinetics were well understood long before the structure AdipoQ, a protein secreted exclusively from adipocytes,
was determined [5]. The signaling function of tyrosine and related to the C1q family of innate immunity proteins,btb107.qxd 02/16/2000 03:20 Page 32
32 Analytical biotechnology
Figure 1
An example of unexpected similarity between proteins with identical in all five proteins are shaded red, and those that are
undetectable sequence homology, the adipocyte secreted factor conserved in four of the five are shaded blue. β-Strand regions are
AdipoQ (Acrp30) and TNFs [11•]. (a) A ribbon diagram comparison of indicated for AdipoQ by green arrows above the sequence alignment,
the AdipoQ and TNFα trimers. (b) Superposition of Cα traces from and below the alignment in blue for CD40L. Sequence identity
monomers of AdipoQ (white) and CD40 ligand, a TNF family cytokine between C1q-like domains and TNFs is negligible (e.g. 9% between
(PDB entry 1ALY). The level of structural similarity is on a par with that AdipoQ and TNFα, with the alignment shown here). Sequence-based
between different TNFs. Note that the corresponding sidechains searches such as BLAST have proved unable to identify relatedness
shown take on remarkably similar orientations and thus conserve the between C1q and TNF family members; however, using residue
overall packing of the cores in the two molecules. (c) Structure-based patterns identified in the structure-based alignment enables the
sequence alignment between the TNFs CD40L, TNFα, and TNFβ and identification of potential new TNF/C1q family members.
the C1q-like domains from C1qA and AdipoQ. Residues that are
which at the time were of unknown structure [11•]. AdipoQ existence of a TNF superfamily, and resulted in the identi-
was discovered in a high-throughput sequencing screen of fication of new members of this class of cytokine. All this
cDNAs enriched in adipocytes. The crystal structure of from determining the crystal structure of one (well-chosen)
AdipoQ revealed clear structural similarity to the TNF protein of unknown function.
family of cytokines (Figure 1). This led to the hypothesis
that AdipoQ might function as a cell signaling factor, which Several important cases of deriving functional hypotheses
now appears to be correct. The similarity to TNF family from structural similarity have arisen in the course of ‘clas-
proteins was not and could not have been found without sical’ structural biology studies. Good examples of these
the structural data. In this case, one key crystal structure include proteins involved in phosphotyrosine binding.
transformed our understanding of the biology of two pro- Several different types of protein domains are known to
tein families (C1qs and TNFs) and their role in the function in binding to phosphotyrosine, including src
evolution of the immune system, has demonstrated the homology-2 (SH2) and phosphotyrosine binding (PTB)btb107.qxd 02/16/2000 03:20 Page 33
Finding function through structural genomics Shapiro and Harris 33
domains, which are not structurally related. When the the very nature of their identification, and structural data
structure of the first PTB domain was determined [12], the may provide key information for deducing their functions.
surprising discovery was made that although being of
apparently unrelated sequence these proteins were struc- Even when applied to proteins with no clear functional
turally similar to pleckstrin homology (PH) domains, which connections, 3D structure determination may allow identi-
bind negatively charged phospholipids to aid in membrane fication of potential binding motifs and catalytic centers.
localization of some proteins. The authors formed the Clearly, this is sometimes difficult, but it has been suc-
hypothesis that PTB domains might serve a similar func- cessfully demonstrated in a number of cases. A good
tion, and then demonstrated through biochemical and example is for the histidine triad (HIT) family of proteins
molecular biological experiments that phospholipid bind- [16–18]. HIT proteins, which were identified through
ing is in fact a natural function of these domains. genomic analysis, form a large and ubiquitous family,
but — as yet — have no known specific biological func-
Structural similarity can sometimes be a very strong indi- tion. Structure solution of several HIT family members
cator of similar function. For example, the crystal structure identified common structural motifs, which were used to
of the amino-terminal domain from the human signaling identify a catalytic center and nucleotide-binding sites.
protein Cbl revealed the presence of a ‘cryptic’ SH2 These structural studies showed that that HIT family pro-
domain [13]. The sequence of this SH2 domain was so teins are nucleotide hydrolases, and even enabled
divergent that its existence had not been previously sus- identification of compounds that could inhibit the catalyt-
pected. The conservation of unique and important ic function of the HIT proteins. Their in vivo substrate is
structural elements, however, clearly identified it as a still unknown. Nonetheless, future biological investigation
member of the SH2 domain family. SH2 domains feature a of this protein family will be fundamentally altered by the
conserved arginine residue that protrudes form an interior knowledge provided by the structure-based functional
position to coordinate the phosphate of bound phospho- genomics approach.
peptides. This arginine is also found the Cbl SH2 domain,
in a structural context that is that is nearly identical to that Structure-based assignment of function can also be aided
of other SH2 domains. These similarities can not be by visualizing cofactors in the electron density of experi-
inferred from sequence comparisons alone. mentally determined structures. For example, Kim and
co-workers [19•] unexpectedly found bound ATP mole-
Clearly, similar structure will not always imply a similar cules in the Methanococcus jannaschii protein MJ0577.
function; but what are the parameters of this relationship? These ATP molecules were scavenged from the Escherichia
Common evolutionary origins might suggest an increased coli expression host, and co-purified with the recombinant-
likelihood of shared functional characteristics — because ly expressed protein. The hypothesis was formed that the
the evolutionary precursors must then have shared a com- molecule might function as an ATPase, and biochemical
mon function. Similar structures alone cannot discern experiments showed that this was in fact correct. In this
evolutionary relatedness in the absence of significant case, the biochemical function could be assigned, but the
sequence identity. Structure-based alignments can some- specific biological function performed by this ATPase is
times reveal conservation of key residues, and other data, still unknown. MJ0577 cannot function as an ATPase in
such as coincidence of intron position and phase, can also isolation, but only in the presence of a soluble extract from
suggest common origins [14]. Nonetheless, functional M. jannaschii. This led the authors to suggest that MJ0577
insights based on structural relationships can only rise to is a factor-dependent ATPase that could potentially func-
the level of hypotheses, and these hypotheses must be tion as a molecular switch in an as yet unidentified cellular
tested by direct functional experimentation. process. Again, we see that the structure-based functional
genomics approach can lead to hypotheses that can be test-
Functional insight from analysis of novel ed by simple biochemical experiments. We cannot expect
protein structures this approach to solve the entirety of most functional prob-
The best — and most famous — example of structural lems, but it may often provide important clues to guide
insight leading to functional knowledge comes not from further analysis. The assignment of a specific functionality
the structure of a protein, but of DNA. Watson’s and to a protein, such as the ATPase activity of MJ0577, sug-
Crick’s famous discovery of the double helix led to the gests that similar functionality will persist in other proteins
greatest biological insight of our era: it did not escape their that are related in primary sequence. Thus, this knowledge
notice that the structure ‘immediately suggests a possible propagates through the database, and may add key infor-
copying mechanism for the genetic material’ [15]. Of mation to other problems as well.
course it was known at the time that DNA was the genet-
ic material, so this may not be a good analogy for many of Other types of biological studies often provide functional
the potential targets presented by high-throughput screen- links between proteins — for example, genetic epistasis
ing. Many proteins identified through genetic techniques, analysis can suggest the connections and order of interac-
positional cloning, and differential expression analysis are, tions in a signaling pathway. Thus, understanding the
however, implicated in a biological or disease process by function of one of these proteins has the potential to rever-btb107.qxd 02/16/2000 03:20 Page 34
34 Analytical biotechnology
berate throughout the entire chain. In one recent real- Genome sequencing projects are also underway for sever-
world example, a few key crystal structures have strongly al species of plants, including major food crops such as rice,
affected understanding of the control mechanisms of maize, tomato, corn, and potato (see http://www.tigr.org).
clathrin-mediated endocytosis [20]. As we begin to understand the functions of more genes
from these species, the prospects for producing new types
of genetically modified strains will increase accordingly
Picking targets wisely may efficiently lead to
[23]. Also, understanding new mechanisms of plant growth
medical and agricultural benefits
control and pathways of natural immunity might lead to
Many disease genes and genes important in biological
new plant husbandry improvements that do not depend on
processes have been identified through methods that impli-
genetic alteration.
cate them in specific functions. For example, positional
cloning can demonstrate that mutation of a particular gene
results in a disease phenotype. This does not, however, Conclusion: what the future may hold
necessarily provide information on the biochemical func- The examples that we have given in this review come
tion of the encoded protein. To rationally develop mainly from ‘classical’ structural biology studies.
therapeutics requires this functional knowledge, and thus Currently, there are several projects underway — both
such proteins are ideal targets for analysis by structure- commercial and academic — to begin structure determi-
based functional genomics. New efforts in nation projects on a genome-wide scale, but the full
pharmacogenomics, particularly those relying on single impact of these efforts probably won’t be felt for the next
nucleotide polymorphism (SNP) data [21], also stand to year or two [24•]. These projects, in addition to their scale,
reap tremendous benefits from a structural treatment. are non-classical in that targets are not chosen based on
Effects of SNPs within protein-coding regions surely derive their known biological interest, but rather are often based
from structural changes in protein structure or functionali- on a lack of specific biological knowledge. These struc-
ty, and these can be fully understood only in the light of tural genomics projects have several different aims,
high-resolution structures. The functional consequences of among them to construct an extensive map of the folds
many gene alterations can be implied by mapping them to that natural proteins adopt. Structure-based functional
the 3D structure of the encoded protein. A good example is genomics will grow out of these efforts, whether or not it
glucokinase, an enzyme involved in the first steps of glu- is their primary mission.
cose metabolism. Mutations in the gene were found to be
co-inherited with a form of maturity onset diabetes in the Although the number of crystal and NMR structures
young (MODY). Biochemical analysis shows that the muta- deposited each year in the protein data bank (PDB;
tions affect the Km and Vmax of the enzyme, and structural http://www.rcsb.org) has grown exponentially, the number
studies show that these mutations map to amino acids lin- of structures unrelated to other proteins has not [24•]. This
ing the active site. Another example is the p53 protein, is due in part to the practice of structural biologists looking
which is involved in many human cancers. A majority of ever deeper at biological problems, and thus studying com-
cancer-causing mutations in p53 map to the region of the plexes, mutants, and relatives of proteins of already known
protein involved in DNA binding [22]. These observations structure. Also, the universe of protein structures is finite,
are entirely dependent on 3D structural knowledge, and it and chance occurrences of structural similarity have
is clear that they have strong implications for our under- become increasingly common. This latter trend would
standing of the underlying disease. appear to be strongly positive for the task of deriving func-
tion from these structures.
Analysis of proteins from pathogens may also provide a
unique opportunity for the application of this technique. Functional genomics approaches in general give correla-
As of October 1999, the complete sequences of 24 micro- tive and not definitive data about the function of new
bial genomes had been determined, and sequencing genes. Hybridization array experiments, for example,
projects on about double that number were underway (see: might demonstrate that a particular gene (and thus its pro-
http://www.tigr.org). In addition, comprehensive ‘knock- tein product) is expressed in step with the cell cycle,
out’ projects have been initiated to identify those genes implying a possible role as a ‘cell-cycle gene’. Genetic
that are essential for viability. Mapping the functions of approaches, hypothetically, might give confirmatory data
these essential genes is an important first step in applying showing that mutations in the gene affect the cell cycle.
this information toward the development of new anti- Neither of these experiments, however, uncover the pro-
microbial drugs. The availability of high-resolution tein’s biochemical role. When the database of protein
structures also adds important data for identifying new structures (with functions proscribed) is large enough, the
drug targets. Whereas a particular protein may be of vital biochemical function of any protein with a 3D structure
importance to the life cycle of a microbe, the best drug tar- can be implied and tested. This ‘function by structure’
gets are usually enzymes with active sites that can be model of genomics will become easier and more rewarding
targeted for therapeutic intervention. This knowledge as time goes on, and the public domain (PDB) and com-
comes as a by-product of structural studies, and can be mercial protein data banks increase in size and
used in the selection of targets for further investigation. comprehensiveness.btb107.qxd 02/16/2000 03:20 Page 35
Finding function through structural genomics Shapiro and Harris 35
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to X-ray crystallographic analysis. The structure revealed similarity to TNF- An excellent review describing the current state of pilot structural genomics
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function as a cytokine. required for structural genomics work.You can also read
