Medical Microbiology and Immunology (2020) 209:265–275

https://doi.org/10.1007/s00430-020-00663-5

REVIEW

Structural proteomics, electron cryo‑microscopy and structural

modeling approaches in bacteria–human protein interactions
Sounak Chowdhury1 · Lotta Happonen1 · Hamed Khakzad2,3,4 · Lars Malmström1,2,3,4 · Johan Malmström1

Received: 13 August 2019 / Accepted: 30 January 2020 / Published online: 19 February 2020
© The Author(s) 2020

Abstract
A central challenge in infection medicine is to determine the structure and function of host–pathogen protein–protein interac-
tions to understand how these interactions facilitate bacterial adhesion, dissemination and survival. In this review, we focus
on proteomics, electron cryo-microscopy and structural modeling to showcase instances where affinity-purification (AP) and
cross-linking (XL) mass spectrometry (MS) has advanced our understanding of host–pathogen interactions. We highlight
cases where XL-MS in combination with structural modeling has provided insight into the quaternary structure of interspe-
cies protein complexes. We further exemplify how electron cryo-tomography has been used to visualize bacterial–human
interactions during attachment and infection. Lastly, we discuss how AP-MS, XL-MS and electron cryo-microscopy and
-tomography together with structural modeling approaches can be used in future studies to broaden our knowledge regarding
the function, dynamics and evolution of such interactions. This knowledge will be of relevance for future drug and vaccine
development programs.

Keywords Host–pathogen interaction · Proteomics · Affinity-purification mass spectrometry · Cross-linking mass

spectrometry · Electron cryo-microscopy · Molecular modeling

Introduction (WHO) have identified infectious diseases and the increas-

ing resistance to antibiotics as a foremost global concern
Infectious diseases are a serious health problem aggravated [3]. The increasing bacterial resistance to antibiotics threat-
by the current spread of pathogens and their vectors to new ens to make some infections untreatable and poses a major
niches. Concomitantly, the emergence of novel, zoonotic threat to modern health care as several medical procedures
pathogens is rapidly increasing as well as the bacterial resist- are dependent on effective antibiotics. Actions are needed to
ance to antibiotics [1, 2]. In fact, several of the major health promote the understanding of the molecular mechanisms by
organizations including the World Health Organization which pathogens cause disease and how they modulate their
host’s cellular machinery to escape immune surveillance.
Edited by Volkhard A. J. Kempf. Equally important is the development of new treatment alter-
natives to antibiotics [4].
This article is published as part of the Special Issue on “Vibrant During an infection, a bacterial pathogen circumvents
ITN”.
host’s immune defenses via highly evolved effector proteins
* Johan Malmström or virulence factors that can hijack and re-wire molecular
[email protected] host systems. At the same time, host proteins such as immu-
noglobulins, proteins of the complement system and antimi-
1
Division of Infection Medicine, Department of Clinical crobial proteins together with cells from the host’s adaptive
Sciences, Faculty of Medicine, Lund University,
22184 Lund, Sweden and innate immune system, bind to bacterial surfaces and
2 effector proteins to neutralize bacteria and prevent infection.
Institute for Computational Science, University of Zurich,
8057 Zurich, Switzerland This dynamic interplay between host and pathogen partly
3 depends on the formation of host–pathogen protein–pro-
Swiss Institute of Bioinformatics (SIB), 1015 Lausanne,
Switzerland tein interactions (HP-PPIs) [5, 6]. Typically, HP-PPIs are
4 dynamic and are under strong evolutionary pressure [7] to
S3IT, University of Zurich, 8057 Zurich, Switzerland

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266 Medical Microbiology and Immunology (2020) 209:265–275

a point where the diversity can inundate the host immune interactions. In this context, highly resolved information of
system [8, 9]. Structural characterization of HP-PPIs has protein binding interfaces across species-specific HP-PPI
the potential to advance our understanding of the molecular networks has the potential to uncover underlying evolu-
mechanisms of infection as well as to provide new targets tionary conserved interaction patterns that can be further
for drug and vaccine development [10, 11]. Integrative struc- exploited for the development of new therapeutic strategies
tural biology combining information from several comple- [22]. Lastly, structural information of the individual pro-
mentary approaches within structural biology and biological tein components as well as intact interspecies protein com-
mass spectrometry has the potential to improve the charac- plexes is required to determine the structural–functional
terization of HP-PPIs (see “INFO BOX 1”) [12]. relationship of the interactions. The recent development
and application of several mass spectrometry (MS)-based
Info box 1: Integrative structural biology protein interaction analysis strategies [23] together with
Integrative structural biology refers to the determination of structural the ‘resolution revolution’ of cryoEM [24–26] offers new
model of a protein or protein complex using a variety of differ- possibilities to map, characterize and functionally annotate
ent structural methods, typically X-ray crystallography, nuclear
magnetic resonance spectrometry (NMR), single-particle electron
HP-PPI networks. In this review, we highlight some recent
cryo-microscopy (cryoEM) and small-angle X-ray scattering [12, technological developments in affinity-purification (AP)
13]. Traditionally, this has meant the fitting of high-resolution crys- and cross-linking (XL) mass spectrometry (MS) applied
tal structures into nanometer resolution cryoEM maps to generate together with electron cryo-tomography (cryoET) to dem-
an atomic model for a larger protein complex. Recent technological
and computational developments are pushing these boundaries, and
onstrate how these approaches have provided novel and
current methods such as chromatographic co-purification of protein distinct information of bacteria–human HP-PPI networks
complexes, cross-linking and hydrogen–deuterium exchange (Fig. 1). We also propose how the increasing maturity of
mass spectrometry, light scattering, mutagenesis and molecular AP-MS, XL-MS, single-particle cryoEM and cryoET is
modeling are adding valuable information for data interpretation
[12, 14]. The individual pieces of data gathered using the different
likely to advance integrative structural biology and mod-
methods provide valuable restraints for the determination of the eling approaches.
conformation(s), position and orientation of the components. The
simultaneous use of such restraints can significantly improve the
accuracy, precision and completeness of a given protein complex
model [12].
Affinity‑purification mass spectrometry
Successful integrative structural biology approaches for
the characterization of HP-PPI networks typically requires AP-MS is an increasingly important technique to explore
multi-tiered information. For example, large-scale map- HP-PPIs based on affinity-tagged bacterial or human pro-
ping of binary interspecies protein–protein interactions teins coupled to a solid matrix to capture interacting pro-
is important to outline the degree of interconnectivity teins (Fig. 2 and “INFO BOX 2”). AP-MS enables the
between proteins within a network. Conversely, generation identification and quantification of multiple proteins that
of multiple HP-PPI networks between different species are enriched during the affinity purification. This technique
will support comparative studies to promote generalized generates information on interspecies protein–protein
conclusions about pathogen-specific mechanisms [15–17] interactions and on occasion the dynamics of such inter-
and common themes of interaction between different actions. In the broadest application, the entire proteome
pathogen types [6, 18–21]. Such information will clarify of a given pathogen can be analyzed—most often that of a
whether particular bacterial proteins bind to more than one virus—by expressing every protein as individual recom-
human protein to form larger host–pathogen protein–pro- binant, affinity-tagged protein to probe proteome-wide
tein complexes (HP-PPC); or if certain human proteins interspecies HP-PPI [27]. Different versions of AP-MS
are frequently targeted by several proteins from one or typically rely on different data acquisition schemas and
many bacterial pathogens [16]. Another important aspect different strategies to filter out false interactions to visual-
is the knowledge of the protein–protein binding site to, for ize the resulting interaction network as highlighted in the
example, differentiate between direct and indirect protein examples below.

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Medical Microbiology and Immunology (2020) 209:265–275 267

Fig. 1  Overview of integra-

tive proteomics, electron Affinity-purification
cryo-microscopy and struc- mass spectrometry
tural modeling approaches in
bacteria–human protein–protein
interactions; HP-PPI host–path- Identification of interacting
ogen protein–protein interac- host proteins Identification on novel
tion, HP-PPC host–pathogen Identification of interacting HP-PPI Bacteria HP-PPCs for cryoEM studies
protein–protein complex, amino acids at HP interface
cryoEM electron cryo-micros-
copy

Cross-linking Electron cryo-

mass spectrometry microscopy
Guided assembly of
multicomponent HP-PPCs
Structural determination of
HP-PPC

Determine quaternary
protein stuctures Infection Drug target identification and
structure driven drug development

medicine

Structural modeling
approaches

A B C D
IDENTIFY NEW HP-PPIs
Relative intensity

SOLID
AP-MS WORKFLOW AFFINITY- TAG BAIT
MATRIX

m/z
PREY PROTEINS
INFECTED HOST-CELLS
HOST-CELL LYSATE ENZYMATIC DIGESTION LC-MS/MS ANALYSIS BIONFORMATIC ANALYSIS &
CONTAMINANTS BAIT PLASMA TO PEPTIDES MOLECULAR MODELING
SALIVA
Relative intensity

m/z-shift

BAIT
XL-MS WORKFLOW CROSS-LINKER

m/z

HP-PPI SUB-COMPLEXES
INTERACTION INTERFACES
PROTEIN STRUCTURE INFORMATION

Fig. 2  Schematic overview of the affinity-purification mass spectrom- ing proteins via either the AP-MS or the XL-MS workflow, all pro-
etry (AP-MS) and cross-linking mass spectrometry (XL-MS) work- teins present in either sample are digested to peptides via dedicated
flows. Interacting prey proteins (e.g., host proteins) to a given bait enzymes, prior to c mass spectrometric analysis of the samples via
(e.g., bacterial protein) can be identified from a variety of biological liquid chromatography tandem mass spectrometry (LC–MS/MS).
mixtures, such as infected cells, host-cell lysates, plasma or saliva In the XL-MS samples, a typical signature feature for a cross-linked
via AP-MS (top panel) or XL-MS (bottom panel). a In the AP-MS peptide is an observable mass-over-charge (m/z) shift in the eluting
workflow, interacting prey proteins are enriched from the biological peptides arising from isotopic variants of the cross-linker molecule.
sample to an affinity-tagged bait protein attached to a solid affinity d Bioinformatic analysis of the acquired spectra allows for the iden-
matrix; whereas in XL-MS, interacting prey proteins can be identi- tification of (novel) HP-PPIs and together with molecular modeling
fied as associated to the bait via adding a suitable cross-linker to the for the identification and structural determination of the HP-PPI sub-
sample and identifying cross-linked bait–prey peptides further down complexes and their interaction interfaces
the workflow. b For the mass spectrometric identification of interact-

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lnc–human interactions. The study identified several proteins

Info box 2: Affinity-purification mass spectrometry (AP-MS)
and pathways known to be modulated during infection and
Affinity-purification mass spectrometry (AP-MS) is based on the
principle of enriching proteins (preys) or other biomolecules from revealed cellular processes possibly modulated by C. tra-
a complex biological mixture using a ligand (the bait) coupled to a chomatis. Importantly, several of these HP-PPIs were found
solid matrix via an affinity-tag (Fig. 2). The bait and the biological to be conserved also in viruses, such as HIV [17, 27].
sample are mixed for the prey proteins to interact and bind to the
These above examples highlight how AP-MS coupled
bait; whereas non-interacting, unbound proteins are washed away.
The bait–prey complexes are subsequently released from the solid with appropriate bioinformatics data analysis strategies can
matrix, enzymatically digested and processed for MS analysis [17, determine interspecies PPIs, provide evidence that several of
28]. The affinity-tagged proteins are often expressed as recombinant the characterized HP-PPI networks are highly interconnected
proteins [17, 28, 29], but in case of intraspecies PPI analysis, they
and that certain bacterial proteins can bind to several human
can equally be expressed by the recombinant cells being investi-
gated [30–32]. Common affinity-tags used include the Strep- [17] proteins to potentially form larger inter-species protein com-
or StrepII-tag [28, 29] and the FLAG-tag [33]. For a comprehen- plexes [16, 17, 28]. One of the challenges with large-scale
sive review of possible tags, see Dunham et al. [34]. The captured AP-MS experiments is to filter out biological meaningful
prey proteins can be identified using various mass spectrometry
interactions from proteins that bind in an unspecific manner
acquisition methods such as data dependent acquisition (DDA) or
more recently data independent acquisition (DIA) and sequential to the bait or the affinity matrix. It can be anticipated, how-
window acquisition of all theoretical mass spectra (SWATH-MS). ever, that the generation of additional HP-PPI networks from
DDA is based on the principle where the most abundant peaks in other species will grant access to ‘‘gold-standard’’ datasets
MS1 spectra within in a fixed time frame are selected to be frag-
of known host–pathogen interactions that can be used to
mented to give rise to MS2 spectra. DIA and SWATH-MS are quite
interlinked where the user defines a set range of m/z and allows optimize a score threshold for removal of false protein inter-
the system to pick peaks within this set range separated by a fixed actions. In this context, novel quantitative MS-based strate-
m/z value to be fragmented. Common data filtering algorithms for gies have already shown to be beneficial for discriminating
distinguishing contaminating proteins from true-positive interactors
between true and false interactions and has furthermore
include, for instance, SAINT [35], ComPASS [36] and MiST [37].
provided new opportunities to reliably quantify temporal
changes of protein interaction networks [38].
In recent work, Happonen et al. generated a quantita-
tive interaction map between the Streptococcus pyogenes
bacterium and human proteins [28]. The map is composed
of over 220 high-confidence HP-PPI between streptococ-
cal virulence factors and human plasma and saliva proteins.
Cross‑linking mass spectrometry (XL‑MS)
The results demonstrated that S. pyogenes forms a highly
Understanding the quaternary structure of molecular
interconnected HP-PPI network with human proteins, which
complexes at a proteome level and close to in vivo condi-
can dynamically change in different bacterial–host micro-
tions holds the potential of improving our understanding
environments. Furthermore, the use of different S. pyo-
of HP-PPIs. Additionally, information regarding protein
genes serotypes and their isogenic mutants revealed that the
interaction sites and direct and indirect protein binding
M1-protein, the main surface-attached virulence factor of
within HP-PPIs provides critically important information
S. pyogenes, interacts with many human proteins forming
to establish the organization and topology of the HP-PPI
a large HP-PPC. These efforts provide relevant information
networks and the arising HP-PPCs [28, 39]. Cross-linking
for future vaccine development programs for S. pyogenes
mass spectrometry (XL-MS) provides valuable informa-
by identifying the localization of opsonizing antibodies to
tion about the structural characteristics of a protein or
specific regions of the M1-protein. In another paper, Penn
protein complex (Fig. 2, and “INFO BOX 3”). The reac-
et al. performed an AP-MS study to identify protein interac-
tive sides of the cross-linker reagents are separated by a
tions formed between secreted Mycobacterium tuberculosis
fixed distance. Thus, identification of cross-linked peptides
(Mtb) proteins and proteins from human macrophages [29].
provides information of the physical proximity of protein
The study generated a global map of 187 high-confidence
secondary structure elements, subunits and domains.
HP-PPI from 34 secreted Mtb proteins. This enabled the
This distance can then be used for molecular modelling of
identification of a specific interaction between the probable
protein complexes [39–41]. Recent improvements in the
conserved lipoprotein LpqN (a secreted Mtb virulence fac-
chemical cross-linking reagents, mass spectrometers and
tor) and the ubiquitin ligase CBL. The identification of the
database search algorithms [42] have improved the analy-
interaction between CBL and LpqN infers a host defense
sis of cross-linked peptides in complex biological samples
mechanism limiting the growth of Mtb in macrophages. In
such as cell lysates [43]. Advanced XL-MS techniques
a third study, Mirrashidi et al. [17] used inclusion membrane
could also play an important role in integrative structural
proteins (Inc) from Chlamydia trachomatis to generate an
biology [14] as highlighted in the examples below.
extensive HP-PPI network composed of 354 high-confidence

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proteins leads to capture of weak or transient interactions

Info box 3: Cross-linking mass spectrometry (XL-MS)
thereby reducing non-specific background [43, 45]. The
Cross-linking mass spectrometry (XL-MS) provides evidence of
proteins interacting with each other by covalently linking MS work using TX-MS revealed that the full structure of the
detectable amino acid pairs together [44] (Fig. 2). Commonly used M1-protein is engaged in protein interactions, as TX-MS
cross-linkers can either be homo-bifunctional linking two identical could confidently locate protein binding interfaces within
amino acids together or hetero-bifunctional linking two differ-
the repeat regions [39]. Interestingly, the model also pro-
ent amino acids together. Different cross-linkers have different
lengths of cross-linker spacer arms between the reactive groups. posed that some of the plasma proteins are interacting with
In the simplest case, the cross-linker is added to the biological other M1-attached human plasma proteins. This observa-
mixture containing the proteins of interest, allowed to react with the tion demonstrates that TX-MS has the capability to deter-
proteins, with subsequent enzymatic digestion of the cross-linked
mine protein interaction site and the ability to distinguish
proteins to peptides followed by MS analysis (Fig. 2). Importantly,
cross-linking can equally well be applied to AP-MS samples during between direct and indirect protein binding [39]. A highly
the experimental setup, cross-linking bait and prey proteins. As in interesting prospect of additional XL-MS studies is the
the case of AP-MS (see “INFO BOX 2”), different MS acquisition comparative HP-PPI network protein binding site analysis
techniques can also be used to acquire information on cross-linked
across species. Such a comparative study could uncover
peptide pairs. Current state-of-the-art data analysis pipelines and
software for XL-MS data analysis (recently reviewed by Yu and underlying evolutionary conserved interaction patterns
Huang [46]), include the TX-MS pipeline [39], the MeroX [47] and [22]. Still, it is clear that additional work is required to
Mass Spec Studio (https​://www.msstu​dio.ca/) software. further address the quadratic expansion of the computa-
tional search space and the unequal fragmentation effi-
In a study by Schweppe et al. [48], cross-linking Acine- ciency of two cross-linked peptides, which typically makes
tobacter baumannii cells with human lung epithelial cells it difficult to unambiguously identify the XL-peptides in
led to the first large-scale HP-PPI analysis for A. baumannii. complex samples. These developments would allow to
Biotin-aspartate proline-PIR n-hydroxyphthalimide (BDP- more routinely integrate XL-MS workflows in integrative
NHP) was used to crosslink A. baumannii with human cells, structural biology approaches.
followed by tryptic digestion of the cross-linked proteins
and subsequent MS analysis. This cross-linking experiment
using human lung epithelial cells identified the outer mem-
brane protein A (OmpA) as a virulence factor. OmpA was Cryo‑electron microscopy to study bacteria–
found to bind to a desmosomal protein providing evidence human interactions
for a novel mechanism of how A. baumannii enables epithe-
lial intrusion and cell invasion. In more recent work, Hauri As detailed above, AP-MS and XL-MS can provide
et al. developed a novel XL-MS workflow termed targeted detailed information of global HP-PPIs, their composi-
chemical cross-linking (TX-MS). TX-MS relies on a com- tion, dynamic regulation, overall topology and specific
bination of chemical cross-linking, high-resolution mass protein–protein interaction sites. However, even when
spectrometry and high-accuracy protein structure modeling combined with structural modeling, XL-MS and AP-MS
[39]. TX-MS was used to construct a high-resolution inter- are unable to provide high-quality, atomic resolution struc-
species quaternary model of the S. pyogenes M1–human tural information of the individual proteins in a complex or
protein complex identified in a previous AP-MS study [28], the structure of the complete HP-PPC. For studies where
as discussed briefly above. The model explains how the high-resolution structural information is required, XL-MS
repeat regions of the streptococcal M1-protein bind to sev- can be combined with single-particle cryoEM (see “INFO
eral plasma proteins along its length to prevent phagocytosis, BOX 4”) as described for several large, multicomponent
inhibit complement activation and to secure nutrients [49]. human protein complexes as recently reviewed [41, 51]. To
At the same time, the model also explains how S. pyogenes date, the majority of studies on HP-PPCs applying either
masks its conserved and vulnerable surface epitopes in the single-particle cryoEM or cryoET have been performed
binding interfaces with human proteins [50]. mostly on virus–host PPCs and PPIs—such as interac-
The above instances demonstrate that XL-MS is a tions with host receptors [52–55]. Such studies show the
promising technique that promotes the analysis of HP- potential for equal ones on bacteria–human interspecies
PPI networks and elucidates the arising protein complex PPCs targeting structures at the host–pathogen interface.
structures. Addition of cross-linkers to proteins stabilizes For bacteria–human HP-PPIs, the examples on electron
their interactions in native conditions, thus providing valu- cryo-microscopy only include cryoET, which, regardless
able information on their dynamics and flexible regions. of being a powerful visualization technique, does not pro-
Moreover, covalent bond formation between interacting vide atomic resolution detail on the PPI interface.

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a host-free environment and in contact with host plasma

Info box 4: Single-particle electron cryo-microscopy and electron
cryo-tomography membrane followed by sub-tomogram averaging, discern-
In single-particle electron cryo-microscopy (cryoEM), the purified ing several conformational differences between these two
protein or protein complex is preserved in vitreous water on sample states. Nans et al., thus, revealed that the T3SS acts like
grids allowing for their native structural state to be maintained [26]. a ‘molecular syringe’ during effector protein release into
Imaging of such samples is performed under cryogenic tempera-
the host-cell cytoplasm [66]. Jasnin et al. used the same
tures to protect the specimen from radiation damage. Here, the
assumption is that the particles studied obtain random orienta- approach by cultivating epithelial kidney cells directly on
tions on the sample grid. During imaging, tens of thousands up the sample grids, infecting them with L. monocytogenes
to million(s) of two-dimensional (2D) projections of individual and visualizing the infected cells by cryoET, followed by
particles are collected [56, 57]. These 2D projections are aligned
tomogram interpretation by an automatic segmentation
and averaged to generate a three-dimensional (3D) reconstruction
of the protein or protein complex using dedicated image-processing algorithm developed specifically for the tracking of actin
algorithms [58, 59]. In cases where the 3D structure reaches atomic filaments [70]. The work by Jasnin et al. proposed a model
resolution, the amino acid sequence can be built into the 3D map of actin nucleation and comet tail assembly on the bacte-
to generate a 3D model of the protein or protein complex. Single-
rial surface with the bacterial ActA and the human Arp2/3
particle cryoEM is typically applied for macromolecular complexes
ranging in size from below 100 kDa (such as hemoglobin [60]) to [71] being the key players, leading to simultaneous polym-
MDa (such as intact viruses [61]). erization of multiple tangential actin filaments [67, 72].
In contrast, electron cryo-tomography (cryoET) allows for the three- Although cryoET is an important visualization tech-
dimensional visualization of intact cells and cellular structures. nique as demonstrated above, it does not provide molecu-
The sample(s) studied (intact cells, larger viruses) is preserved
lar level detail of the HP-PPIs or HP-PPCs. Sub-tomogram
in vitreous water on special sample grids allowing for its native
structural state to be maintained, much like in single-particle averaging of frequent protein–protein contacts during
cryoEM. However, during imaging, the sample is rotated within infection could alleviate this gap in knowledge, much as
the microscope by tilting the grid along one; sometimes two axes, has been done for the C. trachomatis T3SS needle syringe-
and a ‘tilt-series’ of two-dimensional (2D) projections are acquired
like movement when injecting effector proteins into the
and then used for the calculation of a three-dimensional (3D)
reconstruction or tomogram [62]. Due to this imaging technique host cytoplasm [66] or in combination with quantitative
and the lack of single-particle averaging for a higher signal–noise proteomics [65]. There is still considerable amount of
ratio, the achievable resolution is limited. The resolution is further detailed knowledge in infection medicine to be derived
limited by the thickness of the sample, as the electron beam typi-
from such experiments by exploring new bacteria–human
cally can only penetrate 500 nm into the sample [62]. This means
in practice that only prokaryotes can be imaged in toto, whereas host–pathogen systems.
other cells must be thinned down [63]. Mammalian cells and tissues
are often sliced into thinner sections via cryo-sectioning or focused
ion beam milling before visualization [25, 64]. The resolution of
certain symmetric and repetitive features in the tomogram—such
as smaller cellular components or viral surface proteins—can be Structural modeling approaches
increased by sub-tomogram averaging [65, 66]. Here, these features
are processed as individual protein(s) much like as in single-particle The successful integration of biological mass spectrometry
cryoEM, where 2D projections of individual particles are collected,
with cryoEM and cryoET will be strongly dependent on
aligned and averaged to generate a 3D reconstruction of the protein
or protein complex, with the distinction that in sub-tomogram novel structural molecular modeling approaches. Struc-
averaging, the particles are represented by 3D volumes rather than tural molecular modeling has changed in recent years by
2D projections. the introduction of low-resolution modeling techniques
and fragment-based movers. These techniques use seg-
Much of current state-of-the art work in imagining ments of known protein structures to ensure that perturba-
interactions between bacteria and human via cryoET have tions to the model simulation adhere to biochemical con-
been done on Listeria monocytogenes [67] and the intra- straints that determines the three-dimensional structure of
cellular Chlamydia trachomatis [66, 68, 69]. For example, the protein [71] (see “INFO BOX 5”). This revolution and
Nans et al. [68] cultivated human cells directly on sam- the increase of understanding it provides have supported
ple grids, infected them with C. trachomatis elementary the design of enzymes [73], the creation of new protein
bodies (EBs) released naturally from co-cultured infected topologies [74] and the design of self-assembling pro-
cells and visualized them by cryoET. In this way, a sys- teins [75]. Protein–protein docking and flexible-backbone
tem was developed to visualize snapshots of Chlamydial protein–protein docking can now be carried out routinely
EBs under physiological conditions during early stage cell as long as experimental structures or high-quality homol-
entry, including the type III secretion system (T3SS) nee- ogy models exist [76]. Additionally, molecular modeling
dles in direct contact with the host plasma membrane [68]. in conjunction with XL-MS has allowed us to provide
Nans et al. also determined the structure of the T3SS in detailed structures of bacteria–human HP-PPCs [39].

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identifies interacting protein pairs; however, it fails to iden-

Info box 5: Molecular modeling
tify the interacting peptides and domains between proteins.
The first step in modeling the structure of a protein is de novo (ab
initio) modeling of the structure from the amino acid sequence AP-MS coupled with XL-MS would not only identify inter-
without prior knowledge about the spatial arrangement of the acting proteins, but also stabilize transient interactions and
amino acids [77]. This approach predicts a protein’s folding based identify amino acid pairs between the interacting proteins.
on physical/chemical principles without making use of explicit
Complementing AP-MS and XL-MS with single-particle
homolog or template structures in contrast to template-based
algorithms. Some successful de novo approaches according to the cryoEM would provide additional structural information
thirteen Critical Assessment of Techniques for Protein Structure on the complete HP-PPC. Both XL-MS and single-particle
Prediction (CASP13) include MULTICOM [78], SWISS-MODEL cryoEM require small sample amounts and both can be
[79], QUARK [80], and Rosetta [81].
applied to heterogeneous samples. For larger protein com-
Predicting the structure of a protein can also be addressed by com-
plexes, where the local resolution in cryoEM maps can vary
parative modeling approaches when there is a suitable template or
homologous structure that can be used to guide the process. Com- considerably with usually highly defined core regions and
parative modeling approaches mainly align the sequence of two (or more poorly resolved densities towards the edges, XL-MS
more) proteins and use the template structure(s) for the similar parts can help in resolving the structure of these edges by provid-
and try to model the gaps by de novo modeling or other fragment-
ing the needed distance constraints between proteins or their
based approaches. In this category, some of the popular softwares
are RosettaCM [82], Modeler [83], HHpred [84], and I-TASSER domains [51]. However, whereas it is difficult to determine
[85]. via XL-MS whether a cross-link between peptides arises
In addition to the aforementioned methods, models or low-resolution from a more or less populated protein (complex) conforma-
experimental data from NMR or X-ray crystallography can be tion, this information can be determined via single-particle
improved and refined by several computational techniques such as
cryoEM by classification particles and derived volumes dur-
loop-modeling approaches like next-generation KIC (NGK) [86],
and DaReUS-Loop [87, 88] or experimental data-based protocols of ing data processing [51, 98]. Other limitations of XL-MS
Rosetta such as RosettaES [89], CS-Rosetta [90], and RosettaNMR relate to possible unequal fragmentation efficiency of two
[91]. cross-linked peptides and identification of sparse networks
of cross-linked distance constraints. Incorporating struc-
tural modeling approaches such as de novo modeling, com-
parative modeling, and protein–protein docking will play
Future directions—towards an integrative an important role in overcoming these limitations. TX-MS
approach in bacteria–human interactions [39] as mentioned above is a successful example of such
combination, as it overcomes the aforementioned limitations
With current state-of-the-art instrumentation, the constantly and enables the structural modeling of large macromolecular
improving data-processing algorithms and bioinformatics assemblies with dense networks of distance constraints. The
tools—particularly in MS and electron microscopy—there usefulness of XL-MS to structural, proteome-wide studies
is a vast possibility of combining quantitative and struc- will further be pushed by recent developments in cross-link-
tural mass spectrometry with advanced structural biology. ers [43] and structural modeling and docking approaches
Such methods could encompass AP-MS, XL-MS, hydro- [14, 39, 43]. It can further be anticipated that integrative
gen–deuterium exchange mass spectrometry (HDX-MS), structural approaches in medical microbiology will be
native mass spectrometry, single-particle cryoEM, X-ray beneficial for the design of novel therapeutic approaches.
crystallography, NMR, small angle X-ray and neutron scat- For example, the emerging field of structure-based design
tering methods, together with cellular visualization meth- of vaccines—also referred to as structural vaccinology—
ods (foremost cryoET but also, e.g., correlative light and has recently started to deliver new vaccine antigens [99].
electron microscopy). As mentioned earlier, such integrative Structural vaccinology aims at optimizing protective B-cell
studies would advance our understanding in pathogenesis, epitopes using a combination of X-ray, electron microscopy,
yet a lot remains to be done with respect to applying such mass spectrometry and computational approaches [100].
approaches in medical microbiology. Current studies have Pioneering studies have shown that HP-PPI between human
combined electron cryo-microscopy, XL-MS and structural proteins and bacterial proteins influences protective antibody
modeling to a large extent to understand intra-species, i.e., responses [101, 102], which is of importance for rational
human–human protein complexes [40, 92–97]. design of cross-protective antigens [103]. Furthermore, effi-
In this review, we have showcased different techniques cient physical and structural epitope mapping abilities using
involved in understanding different tiers in HP-PPI. Inte- for example XL-MS, HDX-MS, X-ray crystallography and
grative approaches involving AP-MS, XL-MS, cryoEM and more recently cryoEM for large antigens provides essential
structural modelling could provide combined knowledge information for antigen engineering to guide vaccine design
and valuable insights into the process of infection. AP-MS and optimization [100]. Such information in combination
with human B-cell repertoire sequence analysis represents

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