Uromodulin (UMOD)/Tamm-Horsfall protein, the most abundant human urinary protein, plays a key role in chronic kidney diseases and is a promising therapeutic target for hypertension. Via its bipartite zona pellucida module (ZP-N/ZP-C), UMOD forms extracellular filaments that regulate kidney electrolyte balance and innate immunity, as well as protect against renal stones. Moreover, saltdependent aggregation of UMOD filaments in the urine generates a soluble molecular net that captures uropathogenic bacteria and facilitates their clearance. Despite the functional importance of its homopolymers, no structural information is available on UMOD and how it self-assembles into filaments. Here, we report the crystal structures of polymerization regions of human UMOD and mouse ZP2, an essential sperm receptor protein that is structurally related to UMOD but forms heteropolymers. The structure of UMOD reveals that an extensive hydrophobic interface mediates ZP-N domain homodimerization. This arrangement is required for filament formation and is directed by an ordered ZP-N/ZP-C linker that is not observed in ZP2 but is conserved in the sequence of deafness/Crohn's disease-associated homopolymeric glycoproteins α-tectorin (TECTA) and glycoprotein 2 (GP2). Our data provide an example of how interdomain linker plasticity can modulate the function of structurally similar multidomain proteins. Moreover, the architecture of UMOD rationalizes numerous pathogenic mutations in both UMOD and TECTA genes.uromodulin | ZP2 | polymerization | zona pellucida domain | X-ray crystallography U romodulin (UMOD) is expressed in the thick ascending limb of Henle's loop as a GPI membrane-anchored precursor that consists of three EGF-like domains, a domain of unknown function (D8C), and a zona pellucida (ZP) module (1, 2) (Fig. 1A, Top). The latter, containing Ig-like domains ZP-N and ZP-C (3-5), is found in other medically important human glycoproteins linked to infertility (egg coat components ZP1-ZP4), nonsyndromic deafness [inner ear α-and β-tectorin (TECTA/B)], Crohn's disease [glycoprotein 2 (GP2)], and cancer [TGF-β coreceptors betaglycan (BG) and endoglin (ENG)] (6, 7). Upon processing by Ser protease hepsin (8) at a consensus cleavage site (CCS) C-terminal to the ZP module (9), UMOD sheds a C-terminal propeptide (CTP) that contains a polymerization-blocking external hydrophobic patch (EHP), exposing an internal hydrophobic patch (IHP). This event triggers homopolymerization into filaments that are excreted into the urine (4, 10), where UMOD performs a plethora of biological functions, including protection against urinary tract infections, prevention of kidney stones, and activation of innate immunity (1,2,11,12).Although UMOD activity is strictly linked to its supramolecular state (2), the mechanism of ZP module-dependent assembly remains unclear. Mass spectroscopy (MS) analysis of ZP-C disulfide linkages suggests that there are two types of ZP modules with different structures (13). Type II contains 10 conserved Cys (C 1-7,a,b,8 ) and bo...

SummaryRecognition between sperm and the egg surface marks the beginning of life in all sexually reproducing organisms. This fundamental biological event depends on the species-specific interaction between rapidly evolving counterpart molecules on the gametes. We report biochemical, crystallographic, and mutational studies of domain repeats 1–3 of invertebrate egg coat protein VERL and their interaction with cognate sperm protein lysin. VERL repeats fold like the functionally essential N-terminal repeat of mammalian sperm receptor ZP2, whose structure is also described here. Whereas sequence-divergent repeat 1 does not bind lysin, repeat 3 binds it non-species specifically via a high-affinity, largely hydrophobic interface. Due to its intermediate binding affinity, repeat 2 selectively interacts with lysin from the same species. Exposure of a highly positively charged surface of VERL-bound lysin suggests that complex formation both disrupts the organization of egg coat filaments and triggers their electrostatic repulsion, thereby opening a hole for sperm penetration and fusion.

Mammalian fertilisation begins when sperm interacts with the egg zona pellucida (ZP), whose ZP1 subunit is important for fertility by covalently cross-linking ZP filaments into a three-dimensional matrix. Like ZP4, a structurally-related component absent in the mouse, ZP1 is predicted to contain an N-terminal ZP-N domain of unknown function. Here we report a characterisation of ZP1 proteins carrying mutations from infertile patients, which suggests that, in human, filament cross-linking by ZP1 is crucial to form a stable ZP. We map the function of ZP1 to its ZP-N1 domain and determine crystal structures of ZP-N1 homodimers from a chicken homolog of ZP1. These reveal that ZP filament cross-linking is highly plastic and can be modulated by ZP1 fucosylation and, potentially, zinc sparks. Moreover, we show that ZP4 ZP-N1 forms non-covalent homodimers in chicken but not in human. Together, these data identify human ZP1 cross-links as a promising target for non-hormonal contraception.

Autotransporter proteins are virulence factors associated with a wide variety of diseases caused by pathogenic gram-negative bacteria, and they play a variety of roles in pathogenesis including disabling host defences and mediating colonization. Pertactin, a key component of the whooping cough vaccine, is an autotransporter protein. A large sub-family of the autotransporters carries a trypsin-like protease domain, but these enzymes have different substrates and functions. The unique export process which defines the autotransporter family involves the polypeptide chain C-terminus forming a barrel structure in the bacterial outer membrane, but the role of this barrel in secreting of the N-terminal 'passenger' domain remains very unclear. There are now four published crystal structures of passenger proteins or fragments of them. We have compared these models to catalogue common features and to help predict the structures and functions of other autotransporter proteins such as SepA, which is involved in the pathogenicity of Shigella.

SummaryFertilization, the culminating event in sexual reproduction, occurs when haploid sperm and egg recognize each other and fuse to form a diploid zygote. In mammals this process critically depends on the interaction between Izumo1, a protein exposed on the equatorial segment of acrosome-reacted sperm, and the egg plasma-membrane-anchored receptor Juno 1, 2. The molecular mechanism triggering gamete fusion is unresolved because both Izumo1 and Juno lack sequence similarity to known membrane fusogens. Here we report the crystal structure of Izumo1, which reveals a membrane distal region composed of a four-helix bundle connected to a carboxy-terminal immunoglobulin (Ig)-like domain through a β-hairpin stabilized by disulfide bonds. Remarkably, different regions of Izumo1 display significant structural similarities to two proteins expressed by the invasive sporozoite stage of Plasmodium parasites: SPECT1, which is essential for host cell traversal and hepatocyte invasion [3]; and TRAP, which is necessary for gliding motility and invasion [4]. These observations suggest a link between the molecular mechanisms underlying host cell invasion by the malaria parasite and gamete membrane fusion at fertilization.